Nitrofuran Compounds for the Treatment of Cancer and Angiogenesis

ABSTRACT

The invention is directed to the synthesis and use of nitrofuran compounds, especially Nifurtimox, as medicaments to treat cancer, especially neuroblastoma, and to inhibit angiogenesis. The invention also provides compositions, unit dosage forms, and kits comprising the compounds.

FIELD OF THE INVENTION

The invention relates to the synthesis and use of nitrofuran compounds(for example, Nifurtimox) to treat cancer and inhibit angiogenesis. Theinvention also provides therapeutic compositions and kits comprisingnitrofuran compounds.

BACKGROUND

Cancer is the second leading cause of death in the United States. Due tothe ever increasing aging population in the United States, it isreasonable to expect that rates of cancer incidence will continue togrow. Cancer is currently treated using a variety of modalitiesincluding surgery, radiation therapy and chemotherapy. The choice oftreatments depends upon the type, location and dissemination of thecancer. One of the advantages of surgery and radiation therapy is theability to control to some extent the impact of the therapy, and thus tolimit the toxicity to normal tissues in the body. Chemotherapy isarguably the most appropriate treatment for disseminated cancers such asleukemia and lymphoma as well as metastases. Chemotherapy is generallyadministered systemically and thus toxicity to normal tissues is a majorconcern. Not all tumors, however, respond to chemotherapeutic agents andothers, although initially responsive to chemotherapeutic agents, maydevelop resistance. Thus there is a need for a better understanding ofthe mechanisms underlying the formation and progression of cancer andthe development of resistance to treatment. There is also a need formore effective cancer treatments.

Evidence has accumulated over the past several years to support thehypothesis that angiogenesis promotes the growth and progression ofsolid tumors and leukemias. Angiogenesis favors the transition fromhyperplasia to neoplasia i.e. the passage from a state of cellularmultiplication to a state of uncontrolled proliferation characteristicof tumor cells. Angiogenesis also influences the dissemination of cancercells throughout the entire body eventually leading to metastasisformation.

More recent evidence implicates angiogenesis in the pathogenesis ofdiseases other than cancer. For example, angiogenesis seems to provide aconduit for the entry of inflammatory cells into sites of chronicinflammation (e.g., Crohn's disease and chronic cystitis) and destroyscartilage in rheumatoid arthritis. Angiogenesis also contributes togrowth and hemorrhage of atherosclerotic plaques, leads tointraperitoneal bleeding in endometriosis, and is a cause of blindness.Angiogenesis has also been implicated in the pathogenesis of otherdiseases and as a result the search for effective angiogenesisinhibitors has intensified. Several angiogenesis inhibitors haverecently been discovered and some are currently in clinical trials.

Neuroblastoma is a leading cause of cancer death in children; it is themost common extracranial solid tumor in children, and it carries a poorprognosis. Current treatments of intensive chemotherapy, surgery,radiation and autologous bone marrow transplant are often unsuccessfulleaving the patients uncured, weak, and unable to tolerate more intensetreatment. Currently most children greater than 1 year of age failstandard therapies. Only 30% of these children survive up to 5 yearsafter diagnosis^(1,2). New advancements in treatment strategies aretherefore needed to improve the overall survival rate in neuroblastoma.

SUMMARY OF THE INVENTION

The invention provides methods for the improved synthesis of nitrofurancompounds, as well as methods and compositions for the treatment ofcancers and for inhibiting angiogenesis in mammalian, especially human,subjects. The invention is based in part on the serendipitous discoverythat Nifurtimox, a known nitrofuran compound used as an anti-fungalagent, reduced the size of a neuroblastoma tumor in a patient who wasbeing treated with Nifurtimox for Chagas disease. Nifurtimox was alsofound to inhibit proliferation of neuroblastoma cells in vitro. Theinvention is also based in part on the discovery that Nifurtimoxinhibits angiogenesis.

Nifurtimox belongs to a group of compounds known as nitrofurans (FIG.1). Nitrofurans (including Nifurtimox) are nitroheterocyclic compounds,may of which have biological activity against protozoan and bacterialinfections in mammals.⁴ To date, the nitrofurans have not beeninvestigated for use in the treatment of human cancers because ofobserved toxic effects in veterinary animals. For example,nitrofurazone, a veterinary antimicrobial, was found to cause mammaryand ovarian tumors in animals.⁵

The novel observation that Nifurtimox reduces tumor size, inhibitsproliferation of neuroblastoma cells, and inhibits angiogenesisindicates that Nifurtimox and other nitrofuran compounds can be used totreat cancer and diseases or disorders that are mediated or caused byangiogenesis. Some of these diseases and disorders are recited herein astargets of the therapy.

Thus, in one aspect the invention provides a method for treating amammalian, preferably a human, subject having a cancer. The methodcomprises administering to that subject a nitrofuran compound in aneffective amount to treat the cancer. The cancer may be a metastaticcancer. In preferred embodiments, it may be a solid tumor, for example aneuroblastoma, medulloblastoma, peripheral malignant nerve sheath tumor,ependymoma, craniopharyngioma, astrocytoma (juvenile pilocyticastrocytoma, subependymal giant cell astrocytoma, pleimorphicxanthoastrocytoma, anaplastic astrocytoma, or gliomatosis cerebri),meningioma, germinoma, glioma, mixed glioma, choroid plexus tumor,oligodendroglioma (mixed glioma (e.g., oligoastrocytoma) or anaplasticoligodendroglioma), peripheral neuroectodermal tumor, primitiveneuroectodermal tumor (PNET), CNS lymphoma, pituitary adenoma, orSchwannoma. In some epecially preferred embodiments, the cancer is aneuroblastoma or medulloblastoma. In any embodiment, the subject may befree of other indication calling for treatment with the nitrofuran,i.e., it is not required that the subject also have Chagas disease orsome other condition caused by a microbial infection for example. Themethod may additionally comprise treating the subject with chemotherapy,surgery and/or radiation therapy.

In another aspect, the invention provides a method for inhibitingangiogenesis in a mammalian, preferably a human, subject. The methodcomprises administering to that subject a nitrofuran compound in aneffective amount to inhibit angiogenesis. Thesubject may have a cancer,an ocular disease (for example, macular degeneration, a maculopathy,diabetic retinopathy, or retinopathy of prematurity (retrolentalfibroplasia)), a skin disease (for example, infantile hemangioma,verruca vulgaris, psoriasis, neurofibromatosis, or epidermolysisbullosa), an autoimmune disease (for example, rheumatoid arthritis), agynecologic disease (for example, endometrial polyp, endometriosis,dysfunctional uterine bleeding, ovarian hyperstimulation syndrome,polycystic ovary syndrome (PCO), or preeclempsia), a cardiovasculardisease (for example, coronary artery disease, ischemic cardiomyopathy,myocardial ischemia, arteriosclerosis, atherosclerosis, atheroscleroticplaque neovascularization, arterial occlusive disease, ischemia,ischemic ulcers, ischemic or post-myocardial ischemia revascularization,peripheral vascular diseases, or intermittent claudication), or agastrointestinal disease (for example, Crohn's disease and ulcerativecolitis, Buerger Disease, thromboangiitis obliterans, arteriosclerosisobliterans, ischemic ulcers, multiple sclerosis, idiopathic pulmonaryfibrosis, HIV infection, plantar fasciitis, Von Hippel-Landau Disease,CNS hemangioblastoma, retinal hemangioblastoma, thyroiditis, benignprostatic hypertrophy, glomerulonephritis, ectopic bone formation, orkeloids). The cancer may be biliary tract cancer; bladder cancer; bonecancer; brain or CNS cancer; breast cancer; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; fibromael; cancer of the head and neck; gastric cancer;intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemiaincluding acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymphoid leukemia; liver cancer; lung cancer(e.g. small cell and non-small cell); lymphoma including Hodgkin's andNon-Hodgkin's lymphoma; melanoma; oral cavity cancer (e.g., lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer ofthe respiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; thyroid cancer; uterine cancer; cancer of the urinary system, asarcoma, or a carcinoma. The cancer may be a metastatic cancer. In anyembodiment, the subject may be otherwise free of any indication callingfor treatment with the nitrofuran, for example free of Chagas disease.The method may additionally comprise treating the patient withchemotherapy, surgery and/or radiation therapy.

In another aspect, the invention provides pharmaceutical compositionsfor the treatment of the foregoing diseases, disorders, or conditions.The compositions comprise one or more than one nitrofuran compound inadmixture with a pharmaceutically acceptable carrier. Preferably, thenitrofuran compound is Nifurtimox, Furazolidine or Nifuratel. Morepreferably, the nitrofuran compound is Nifurtimox. In one very specificaspect, the composition comprises a pharmaceutical unit dosage formcomprising an amount of a nitrofuran compound, preferably Niturtimox,effective to treat a neuroblastoma or other related cancer, i.e., acentral nervous system cancer. Preferably the unit dosage is about200-300 mg of medicament, in admixture with a pharmaceuticallyacceptable carrier. In another very specific aspect, the compositionscomprise a nitrofuran compound, especially Nifurtimox, and may alsoinclude ascorbic acid or buthionine sulfoximine as a second activeingredient or agent. These compositions may be formulated for oral,intrathecal, intravenous, or intramuscular administration; oraladministration formulations are preferred.

In yet another aspect, the invention is a kit comprising a nitrofurancompound, for example Nifurtimox, in admixture with a suitablepharmaceutically acceptable carrier, formulated for oral, intrathecal,intravenous, or intramuscular administration. Oral formulation ispreferable. The kit may also include ascorbic acid or buthioninesulfoximine or both in effective amount(s), likewise in suitablepharmaceutically acceptable carrier(s). The kit may further compriseinstructions for use. In some embodiments the instructions for useinstruct the health care provider how to administer Nifurtimox.

As noted above, the compositions, their uses and the kits mayadditionally include a second agent or ingredient. The second agent maybe a glutathione antagonist or depletor. Examples of glutathioneantagonists or depletors include but are not limited to buthioninesulfoximine, isothiocyanates, cyclophosphamide, ifosphamide, actinomycinD, or N-(4-hydroxyphenyl)retinamide (4-HPR). The second agent may be apro-oxidant. Examples of pro-oxidants include but are not limited toascorbic acid, hydrogen peroxide, and hydroquinone. Pro-oxidants areknown to those of ordinary skill in the art. In some importantembodiments the pro-oxidant (e.g., ascorbic acid) is administeredsimultaneously with or before the nitrofuran compound. The second agentmay be a chemotherapeutic agent. Examples of some importantchemotherapeutic agents include but not limited to topotecan,organometallics like cisplatin, paraplatin, doxorubicin, vincristine,vinblastine, taxol and congeners there from, actinomycin D. Exampls ofthese chemotherapeutic agents are listed below. The second agent may bea vascular disrupting agent. Examples of vascular disrupting agentsinclude but are not limited to combretostatins, isothiocyanates bothnaturally occurring or synthetic derivatives and analogs thereof. Thesecond agent may be an angiogenesis inhibitor. Examples of angiogenesisinhibitors include but are not limited to 2-methoxyestradiol (2-ME),AG3340, Angiostatin, Antithrombin III, Anti-VEGF antibody, Batimastat,bevacizumab (avastatin), BMS-275291, CAI, Canstatin, Captopril,Cartilage Derived Inhibitor (CDI), CC-5013, Celecoxib (CELEBREX®),COL-3, Combretastatin, Combretastatin A4 Phosphate, Dalteparin(FRAGIN®), EMD 121974 (Cilengitide), Endostatin, Erlotinib (TARCEVA®),gefitinib (Iressa), Genistein, Halofuginone Hydrobromide(TEMPOSTATINT™), Id1, Id3, IM862, imatinib mesylate, Inducible protein10, Interferon-alpha, Interleukin 12, Lavendustin A, LY317615 or AE-941(NEOVASTATT™), Marimastat, Maspin, Medroxpregesterone Acetate, Meth-1,Meth-2, Neovastat, Osteopontin cleaved product, PEX, Pigment epitheliumgrowth factor (PEGF), Platelet factor 4, Prolactin fragment,Proliferin-related protein (PRP), PTK787/ZK 222584, Recombinant humanplatelet factor 4 (rPF4), Restin, Squalamine, SU5416, SU6668, Suramin,Taxol, Tecogalan, Thalidomide, Thrombospondin, TNP-470, Troponin I,Vasostatin, VEG1, VEGF-Trap, and ZD6474.

The angiogenesis inhibitor may be a VEGF antagonist. In some embodimentsthe VEGF antagonist is a VEGF binding molecule. The VEGF bindingmolecule may be a VEGF antibody or antigen binding fragment thereof. Insome embodiments the VEGF antagonist is NeXstar.

The therapeutic composition of the invention may be administered orally,sublingually, buccally, intranasally, intravenously, intramuscularly,intrathecally, intracranially, intraperitoneally, subcutaneously,intradermally, topically, rectally, vaginally, intrasynovially orintravitreously.

In another aspect, the invention includes an improved method of makingthe nitrofuran compound and nitrofuran analogs of the invention. Thismethod results in a more efficient and less hazardous synthesis of theside chain of nitrofuran and of nitrofuran analogs and is described indetail in the Detailed Description.

These and other aspects of the invention, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription. Each aspect of the inventions can encompass variousembodiments as will be understood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the structure of the backbone of a nitrofuran compound.

FIG. 2A is a histogram showing cell viability of neuroblastoma cells atdifferent doses of nifurtimox. Cell viability assay: SMS-KCN, SMS-KCNR,and IMR-32 cells were cultured in 48 well plates (50,000 cells/well) andtreated with 1 μg/ml, 10 μg/ml or 20 μg/ml of Nifurtimox for 120 hours.Cell viability was assessed using the MTS assay as described inMaterials and Methods and expressed as percent of vehicle treatedcontrol. The data represents the mean±SD of four replicates.

FIG. 2B is a histogram showing BrdU incorporation at different doses ofnifurtimox. Cell proliferation assay: SMS-KCNR cells were cultured in 48well plates and treated with 0, 1.0, 5.0, 10 and 20 μg/ml nifurtimox for48 hours. DNA synthesis was determined by BrdU incorporation assay asdescribed in the Example. Results are expressed as percentages ofuntreated controls and means of six replicates (±SD).

FIG. 3A is a set of photographs showing the effect of nifurtimox onneuroblastoma cells. Sub-confluent cultures of SMS-KCNR cells weretreated with 0, 1.0, 10 and 20 μg/ml nifurtimox for 96 hours. The cellswere photographed using a light microscope as described in Materials andMethods at ×100 magnification. Vehicle treated cells were used ascontrol. Panels i: Vehicle control, ii: 1 μg/ml, iii: 10 μg/ml, and iv:20 μg/ml nifurtimox.

FIG. 3B is a set of pictures showing apoptotic cell death ofneuroblastoma cells by nifurtimox. SMS-KCNR Cells were cultured andincubated with 0, 1.0, 10 and 20 μg/ml of nifurtimox for 96 hours, TUNELassay was performed, photographed and processed as described in theExample. Magnification=100×. i to iv are representative pictures ofoverlaid apoptotic stain and nuclear stain. TUNEL positive nuclei due toDNA fragmentation in SMS-KCNR cells indicates occurrence of apoptoticcell death by nifurtimox treatment. (i): Vehicle control, (ii): 1 μg/ml,(iii): 10 μg/ml, and (iv): 20 μg/ml. The number of apoptotic nucleiincreases with increasing nifurtimox dose.

FIG. 4A is a picture of a slot showing the effect of nifurtimox incaspase-3 activation in neuroblastoma cells. SMS-KCNR cells werecultured and incubated with increasing concentrations of nifurtimox for96 hours. The cells were lysed, separated on 12% SDS PAGE, blotted on toPVDF membrane and probed with antibodies specific for activatedcaspase-3 as described in the Example. The blots were stripped andreprobed with actin antibodies as loading control. Upper panel—ActivatedCaspase-3, Lower panel—Actin.

FIG. 4B is a histogram showing the effect of nifurtimox on neuroblastomacell viability in the absence or presence of Z-VAD-FMK. SMS-KCNR cellswere pretreated with pancaspase inhibitor, Z-VAD-FMK. SMS-KCNR cellswere pretreated with 50 μM Z-VAD-FMK for 90 minutes before treatmentwith nifurtimox (10 μg/ml) for 96 hours. Cell viability was measured byMTS assay. The reversal of cytotoxicity by pancaspase inhibitor wasdetermined by comparing the viability of nifurtimox treated cells in thepresence or absence of the pancaspase inhibitor. The values are themean±SD of quadruplicates.

FIG. 5 is a picture of a Western Blot showing the effect of nifurtimoxon Akt Phosphorylation. (A): SMS-KCNR cells were serum deprived for 18hours, treated with 0, 1.0, 10 and 20 μg/ml nifurtimox for 90 minutesand then stimulated with BDNF (100 μg/ml) for 10 minutes. Cells werelysed and analyzed by western blot analysis using phospho-Akt antibodies(upper panel). The blots were stripped and reprobed with antibodiesspecific for total Akt protein (lower panel). (B): SMS-KCNR cells weretreated with nifurtimox for 90 minutes in the presence of serum. Cellswere then lysed and analyzed by western blot analysis using antibodies.

FIG. 6 is a set of pictures showing the inhibition of angiogenesis bynifurtimox on microvascular sprouting in growth factor stimulated aorticring assay. (A) Vehicle treated aorta without growth factor (B) Vehicletreated aorta with growth factor. (C), (D), (E) represent nifurtimoxtreatment at 1, 10 and 20 ug/ml concentrations in presence of growthfactor.

FIG. 7 is a histogram showing the inhibition of human aortic endothelialcell proliferation by nifurtimox in a dose dependent manner as reflectedby the decreased incorporation of bromo deoxy uridine (BrDu) in the DNAsynthesis.

FIG. 8 (A-C) is a set of pictures showing the inhibition of tubeformation, a critical step in the angiogenesis process by nifurtimox ina dose dependent manner. (A) Vehicle treated endothelial cells withoutgrowth factor (B) Vehicle treated endothelial cells with growth factor.(C) represents nifurtimox treatment at 20 ug/ml concentrations inpresence of growth factor figure and quantitative graph. (D) is ahistogram showing the quantitative analysis of the effect of nifurtimoxon fuse formations assay.

FIG. 9 is a histogram showing the effect of nifurtimox in combinationwith buthionine sulfoximine (BSO) on neuroblastoma cell viability. SMSKCNR cells were cultured overnight and then treated with 0, 1, or 10μg/ml nifurtimox in combination with either 1, 50 or 100 μM BSO for 48hours. Cell viability was measured with Calcein AM assay.

FIG. 10 is a histogram showing the effect of nifurtimox in combinationwith either ascorbic acid or BSO on neuroblastoma cell viability. SY5Ycells were cultured overnight and then treated with either 10 μg/mlnifurtimox (N), 0.3 mM ascorbic acid (A) or 50 μM BSO (B) alone or incombinations (NB, NA, NBA) for 24 hours. Cell viability was measuredwith Calcein AM assay. The results are expressed as a percent of vehiclecontrol.

FIG. 11 is a histogram showing the effect of nifurtimox treatment onneuroblastoma xenograft mice as described in Example 12.

FIG. 12 is a schematic diagram illustrating the scheme for the synthesisof the nitrofuran compounds of the invention as described in detail inExample 16.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the serendipitous discovery (asdetailed in Example 13) that the administration of4-[5-Nitrofurfurylidene)amino]-3-methylthiomorpholine 1,1-dioxide, whichis also referred to by it nonproprietary name “Nifurtimox”, reducestumor size, inhibits proliferation of neuroblastoma cells, and inhibitsangiogenesis. Thus, the invention includes, in some aspects,administering to a subject having a cancer a nitrofuran compound in theform of a medicament to treat the cancer in the subject. The inventionalso includes, in some aspects, administering a nitrofuran compound to asubject to inhibit angiogenesis in the subject. The nitrofuran compoundis administered in an effective and physiologically acceptable amount totreat the cancer in the subject. Although not wishing to be bound to anyparticular theory, we believe that Nifurtimox exerts its cytotoxiceffect specifically by generating free radicals. Nifurtimox is anitroheterocyclic compound; its nitro group can be reduced to the nitroanion radical in cell-free systems by interacting with cytochrome P-450reductase, xanthine oxidase, ascorbate, and catecholamines. Nitro anionscan then reduce oxygen to the superoxide anion radical and hydrogenperoxide. In Chagas disease, the nitro anion free radicals andoxyradicals have been shown to be cytotoxic for the parasite T. Cruzi.The reduction of the nitro group not only generates anion radicals, butinteraction with catecholamines³ appears also to generate semiquinonefree radicals that exacerbate damage to functionally importantbiomolecules, leading to apoptosis of neuroblastoma cell lines.Neuroblastoma cells are known to contain high levels of catecholamines,thereby potentially leading to relatively specific targeting of thesecells. The reaction with catecholamines in neuroblastoma cell lines wasconfirmed by the reduction of cytotoxicity by pretreatment with AMPT, atyrosine hydroxylase inhibitor that reduces the total amount ofcatecholamine stored in cells. In addition, the enhanced sensitivity ofsympathetic neurons—but not parsaympathetic neurons ornon-neuronalcells—to nifurtimox supports this conclusion.

The term “treatment” or “treating” includes amelioration, cure ormaintenance (i.e., the prevention of relapse) of a disorder. Treatmentafter a disorder has started aims to reduce, ameliorate or altogethereliminate the disorder, and/or its associated symptoms, to prevent itfrom becoming worse, or to prevent the disorder from re-occurring onceit has been initially eliminated (i.e., to prevent a relapse).

A subject means a mammalian species, including but not limited to a dog,cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjectscan be house pets (e.g., dogs, cats), agricultural stock animals (e.g.,cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice,rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but arenot so limited. Preferred subjects are human subjects. The human subjectmay be a pediatric, adult or a geriatric subject.

As used herein the terms “nitrofuran(s)” and “nitrofuran compound(s)”are employed interchangeably and encompass furans having a side chaincontaining one or more nitrogen atoms. As is well known in the art,furan is an unsaturated aromatic heterocyclic compound composed of fourcarbon atoms and one oxygen atom. See Ege, Organic Chemistry, 3d. Ed.,D.C. Heath & Co., Lexington, Mass. (1994). Examples of nitrofuransinclude, without limitation, Nifurtimox, Furazolidine and Nifuratel. SeeRaether W, Hanel H. Nitroheterocyclic Drugs with Broad SpectrumAcitivity, Parasit Res 2003; 90:S19-S39; Albrecht et al., J. Med. Chem.13(4): 736 (1970); Albrecht et al., Arzneimittel-Forschung (Drug Res.)21(1): 127-31 (1971); Pozas et al., Bioorganic & Medicinal ChemistryLetters 15: 1417-21 (2005). “Compound” includes both the syntheticallyprepared and administered nitrofuran compound and nitrofuran compoundsproduced in vivo after administration of another compound.

“Cancer” as used herein refers to an uncontrolled growth of cells whichinterferes with the normal functioning of the bodily organs and systems.Cancer cells which migrate from their original location and seed vitalorgans can eventually lead to the death of the subject through thefunctional deterioration of the affected organs. A cancer cell is a cellthat divides and reproduces abnormally due to a loss of normal growthcontrol. Cancer cells almost always arise from at least one geneticmutation. In some instances, it is possible to distinguish cancer cellsfrom their normal counterparts based on profiles of expressed genes andproteins, as well as to the level of their expression. Genes commonlyaffected in cancer cells include oncogenes, such as ras, neu/HER2/erbB,myb, myc and abl, as well as tumor suppressor genes such as p53, Rb,DCC, RET and WT. Cancer-related mutations in some of these genes lead toa decrease in their expression or a complete deletion. In others,mutations cause an increase in expression or the expression of anactivated variant of the normal counterpart.

The term “tumor” is usually equated with neoplasm, which literally means“new growth” and is used interchangeably with “cancer.” A “neoplasticdisorder” is any disorder associated with cell proliferation,specifically with a neoplasm. A “neoplasm” is an abnormal mass of tissuethat persists and proliferates after withdrawal of the carcinogenicfactor that initiated its appearance. There are two types of neoplasms,benign and malignant. Nearly all benign tumors are encapsulated and arenoninvasive; in contrast, malignant tumors are almost never encapsulatedbut invade adjacent tissue by infiltrative destructive growth. Thisinfiltrative growth can be followed by tumor cells implanting at sitesdiscontinuous with the original tumor.

A metastasis is a region of cancer cells, distinct from the primarytumor location resulting from the dissemination of cancer cells from theprimary tumor to other parts of the body. At the time of diagnosis ofthe primary tumor mass, the subject may be monitored for the presence ofmetastases. Metastases are most often detected through the sole orcombined use of magnetic resonance imaging (MRI) scans, computedtomography (CT) scans, blood and platelet counts, liver functionstudies, chest X-rays and bone scans in addition to the monitoring ofspecific symptoms.

The method of the invention can be used to treat cancer in a subject. Insome embodiments, the cancer is a central nervous system (CNS) cancer.Examples of some important CNS cancers include, but are not limited to,neuroblastoma, medulloblastoma, peripheral malignant nerve sheath tumor,ependymoma, chraniopharyngioma, astrocytoma, meningioma, germinoma,glioma, mixed glioma, choroid plexus tumor, oligodendroglioma,peripheral neuroectodermal tumor, primitive neuroectodermal tumor(PNET), CNS lymphoma, pituitary adenoma, and Schwannoma. In someembodiments the astrocytoma is Grade I, Grade II, Grade III, or GradeIV. The astrocytoma may be a low-grade or a high-grade. The astrocytomamay be juvenile pilocytic astrocytoma, subependymal giant cellastrocytoma, pleimorphic xanthoastrocytoma, anaplastic astrocytoma, orgliomatosis cerebri. In some embodiments the oligodendroglioma is amixed glioma (oligoastrocytoma) or an anaplastic oligodendroglioma. Inone preferred embodiment, the cancer is neuroblastoma.

Other cancers that can be treated by the methods of this inventioninclude but are not limited to basal cell carcinoma, biliary tractcancer, bladder cancer, bone cancer, brain and CNS cancer, breastcancer, cervical cancer, choriocarcinoma, colon and rectum cancer,connective tissue cancer, cancer of the digestive system, endometrialcancer, esophageal cancer, eye cancer, fibroma, cancer of the head andneck, gastric cancer, intra-epithelial neoplasm, kidney cancer, larynxcancer, leukemia including acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphoid leukemia, livercancer, lung cancer (e.g. small cell and non-small cell), lymphomaincluding Hodgkin's and Non-Hodgkin's lymphoma, melanoma, oral cavitycancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer,pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma,rectal cancer, renal cancer, cancer of the respiratory system, sarcoma,skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterinecancer, cancer of the urinary system, as well as other carcinomas andsarcomas.

Carcinomas are cancers of epithelial origin. Carcinomas intended fortreatment with the methods of the invention include, but are not limitedto, acinar carcinoma, acinous carcinoma, alveolar adenocarcinoma (alsocalled adenocystic carcinoma, adenomyoepithelioma, cribriform carcinomaand cylindroma), carcinoma adenomatosum, adenocarcinoma, carcinoma ofadrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also calledbronchiolar carcinoma, alveolar cell tumor and pulmonary adenomatosis),basal cell carcinoma, carcinoma basocellulare (also called basaloma, orbasiloma, and hair matrix carcinoma), basaloid carcinoma, basosquamouscell carcinoma, breast carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma (also called cholangioma andcholangiocarcinoma), chorionic carcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epibulbar carcinoma, epidermoid carcinoma,carcinoma epitheliale adenoides, carcinoma exulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,gigantocellulare, glandular carcinoma, granulosa cell carcinoma,hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma(also called hepatoma, malignant hepatoma and hepatocarcinoma), Hürthlecell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantileembryonal carcinoma, carcinoma in situ, intraepidermal carcinoma,intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma mastitoides, carcinomamedullare, medullary carcinoma, carcinoma melanodes, melanoticcarcinoma, mucinous carcinoma, carcinoma muciparum, carcinomamucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucouscarcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, carcinomanigrum, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma,ovarian carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prostate carcinoma, renal cell carcinoma ofkidney (also called adenocarcinoma of kidney and hypernephoroidcarcinoma), reserve cell carcinoma, carcinoma sarcomatodes, scheinderiancarcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cellcarcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma,spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum,squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinomavilosum.

Sarcomas are rare mesenchymal neoplasms that arise in bone and softtissues. Different types of sarcomas are recognized and these include:liposarcomas (including myxoid liposarcomas and pleiomorphicliposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheralnerve sheath tumors (also called malignant schwannomas,neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (includingEwing's sarcoma of bone, extraskeletal (i.e., non-bone) Ewing's sarcoma,and primitive neuroectodermal tumor [PNET]), synovial sarcoma,angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma,hemangioendothelioma, fibrosarcoma, desmoid tumor (also calledaggressive fibromatosis), dermatofibrosarcoma protuberans (DFSP),malignant fibrous histiocytoma (WIFE), hemangiopericytoma, malignantmesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clearcell sarcoma, desmoplastic small cell tumor, gastrointestinal stromaltumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (alsoknown as osteogenic sarcoma)-skeletal and extraskeletal, andchondrosarcoma.

The cancers to be treated may be refractory cancers. As used herein, arefractory cancer is a cancer that is resistant to the ordinary standardof care prescribed. These cancers may appear initially responsive to atreatment and then recur, or they may be completely non-responsive tothe treatment. Subjects being treated according to the invention for arefractory cancer therefore may have already been exposed to anothertreatment for their cancer. Alternatively, if the cancer is likely to berefractory (e.g., given an analysis of the cancer cells or history ofthe subject), then the subject may not have already been exposed toanother treatment. Examples of refractory cancers include but are notlimited to leukemias, melanomas, renal cell carcinomas, colon cancer,liver (hepatic) cancers, pancreatic cancer, Non-Hodgkin's lymphoma, andlung cancer.

The invention can also be used to treat cancers that are immunogenic.Cancers that are immunogenic are cancers that are known to (or likelyto) express immunogens on their surface or upon cell death. Theseimmunogens are in vivo endogenous sources of cancer antigens and theirrelease can be exploited by the methods of the invention in order totreat the cancer. Examples of immunogenic cancers include malignantmelanoma and renal cell cancer. Mantel Cell Lymphoma, follicularlymphoma, diffuse large B-cell lymphoma, T-cell acute lymphoblasticleukemia, Burkitt lymphoma, myeloma, immunocytoma, acute promyelocyticleukemia, chronic myeloid/acute lymphoblastic leukemia, acute leukemia,B-cell acute lymphoblastic leukemia, anaplastic large cell leukemia,myelodysplastic syndrome/acute myeloid leukemia, Non-Hodgkin's lymphoma,chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL).acute myelogenous leukemia (AML), Common (pre-B) acute lymphocyticleukemia, malignant melanoma, T-cell lymphoma, leukemia, B-celllymphoma, epithelial malignancies, lymphoid malignancies, gynecologicalcarcinomas, biliary adenocarcinomas, and ductal adenocarcinomas of thepancreas.

The invention involves in some other aspects, methods for inhibitingangiogenesis in a subject. Angiogenesis is an abnormal rapidproliferation of endothelial cells resulting in persistent and unabatedformation of abnormal new blood vessels (microvessels). Angiogenesisthat continues for months or years can support the growth andprogression of cancer and may result in damage to various organs andtissues such as, for example, the eye, skin, heart, blood vessels, lung,gastrointestinal tract, and the genitourinary tract. The methods of theinvention involve administering to a subject a nitrofuran compound in aneffective amount to inhibit the angiogenesis. The nitrofuran compound isadministered in an effective amount to inhibit the angiogenesis in thesubject. Preferably the compound is Niturtimox, and the subject is ahuman subject.

As used herein the term “inhibits angiogenesis” refers to the reductionof the number or density of the abnormal microvessels. A reduction ofthe number of abnormal microvessels refers to decreasing the number ofexisting abnormal microvessels or decreasing the production of newmicrovessels. Reduction, as used herein, includes total elimination oreradication, as well as other decreases which do not result in totaleradication.

Angiogenesis may be assesed by various methods or techniques. The mostwidely used method in clinical settings relies on histochemical orimmunohistochemical staining of blood vessels (microvessels) in biopsies(open or needle) or specimens. Features of angiogenesis that may beexamined include, for example, blood vessel density and/or themorphology and/or thickness of the perivascular cuff. Areas ofmicrovessel density in a histologic biopsy or specimen are quantified.Areas of high microvessel density (“hot spots”) may, for example,contain the most tumor cells and/or have the highest chance ofmetastasizing. One technique of determining microvessel density is bymeasuring intercapillary distance. Another method of assessingangiogenesis is measuring perivascular cuff thickness. An increase inthe thickness of the preivascular cuff is associated with progression ofthe angiogenesis and may be indicative of disease worsening.

Angiogenesis may also be assessed by measuring blood, serum, plasma, ortissue levels of angiogenesis (angiogenic) factors. Levels of angiogenicfactors serve as a surrogate marker of angiogenesis. Examples ofangiogenic factors that may serve as surrogate markers of angiogenesisinclude but are not limited to Angiogenin, Angiopoietin-1, Del-1,Fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin,Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,Placental growth factor, Platelet-derived endothelial cell growth factor(PD-ECGF), Platelet-derived growth factor-BB (PDGF-BB), Pleiotrophin(PTN), Progranulin, Proliferin, Transforming growth factor-alpha(TGF-alpha), Transforming growth factor-beta (TGF-beta), Tumor necrosisfactor-alpha (TNF-alpha), Vascular endothelial growth factor(VEGF)/vascular permeability factor (VPF). Imaging techniques are alsouseful for the assesment of angiogenesis. Suitable imaging techniques ordevices include non-invasive devices such as CT, rotational CT,micro-CT, multiple energy computed tomography (MECT), single detector CT(SDCT), multi-detector CT (MDCT), volumetric CT (VCT), MRI, micro-MR,X-ray, rotational X-ray, PET, near infrared/optical and othernon-invasive scanning techniques and devices that may be used outside asubject's body or inserted non-invasively into a body cavity.Angiogenesis may also be imaged by CT angiography (CTA), tomosynthesis,X-ray micro-angiography, and by other techniques. One angiogenesisimaging technique involves the use microbubble-based contrast agents(SonoVue) combined with ultrasound and contrast specific imagingmodalities to detect perfusion changes on tumor microvascular perfusion.Other angiogenesis imaging techniques include color Doppler andmammography. Color Doppler imaging can demonstrate angiogenesis intumors such as breast cancer. Mammography may reveal the vascularizedrim of a breast tumor. A wide range of imaging or radiologic signs maybe enhanced by dyes.

Angiogenesis may also be assessed in a subject by a process thatinvolves introducing at least one contrast agent into a body region ofinterest. For example, a contrast agent for detecting blood vessels maybe injected into a blood vessel. A small amount of contrast agent may beintroduced locally to enhance the detection of blood vessels in aparticular body region of interest. Alternatively, a contrast agent maybe provided in an amount sufficient to enhance the detection of bloodvessels in a large body region or in the entire subject body. Structuredata may be obtained for the body of the subject, or may be obtained forone or more target organs e.g., a lung, heart, breast, colon, etc.,portion of an organ, or another target volume of the subject's body. Atarget volume can be any portion of the subject's body. e.g., a limb,the abdomen, the torso, the neck, the head, or any portion thereof.Other methods or techniques to assess angiogenesis not described hereinmay be used for the purpose of this invention. Methods and techniques toassess angiogenesis are known to those of ordinary skill in the art.

The nitrofurans may be administered in combination with other therapies,such as for example radiation therapy, surgery, conventionalchemotherapy or with a combination of one or more additional therapies.

The nitrofurans may be administered alone in a pharmaceuticalcomposition, or combined with therapeutically effective andphysiologically acceptable amounts of one or more other activeingredients or agents. Such other active ingredients include, but arenot limited to, glutathione antagonists, angiogenesis inhibitors,chemotherapeutic agent(s), and antibodies (e.g., cancer antibodies). Thenitrofuran compound and the other active ingredients or agents may beadministered simultaneously or sequentially. When the nitrofurancompound is administered simultaneously with another active agent orcombined with another active ingredient, the nitrofuran compound and theother active ingredient may be administered in the same or separateformulations, but are administered at the same time. The other activeagents may be administered sequentially with one another and with thenitrofuran compound when the administration of the other active agentand the nitrofuran is temporally separated. The separation in timebetween administrations may be a matter of minutes, hour, days, or itmay be longer.

Examples of glutathione antagonists include but are not limited tobuthionine sulfoximine, cyclophosphamide, ifosphamide, actinomycin D,and N-(4-hydroxyphenyl)retinamide (4-HPR).

Examples of angiogenesis inhibitors include but are not limited to2-methoxyestradiol (2-ME), AG3340, Angiostatin, Antithrombin III,Anti-VEGF antibody, Batimastat, bevacizumab (avastatin), BMS-275291,CAI, Canstatin, Captopril, Cartilage Derived Inhibitor (CDI), CC-5013,Celecoxib (CELEBREX®), COL-3, Combretastatin, Combretastatin A4Phosphate, Dalteparin (FRAGIN®), EMD 121974 (Cilengitide), Endostatin,Erlotinib (TARCEVA®), gefitinib (Iressa), Genistein, HalofuginoneHydrobromide (TEMPOSTATINT™), Id1, Id3, IM862, imatinib mesylate,Inducible protein 10, Interferon-alpha, Interleukin 12, Lavendustin A,LY317615 or AE-941 (NEOVASTATT™), Marimastat, Maspin, MedroxpregesteroneAcetate, Meth-1, Meth-2, Neovastat, Osteopontin cleaved product, PEX,Pigment epithelium growth factor (PEGF), Platelet factor 4, Prolactinfragment, Proliferin-related protein (PRP), PTK787/ZK 222584,Recombinant human platelet factor 4 (rPF4), Restin, Squalamine, SU5416,SU6668, Suramin, Taxol, Tecogalan, Thalidomide, Thrombospondin, TNP-470,Troponin I, Vasostatin, VEG1, VEGF-Trap, and ZD6474. In some embodimentsthe angiogenesis inhibitor is a VEGF antagonist. The VEGF antagonist maybe a VEGF binding molecule. VEGF binding molecules includeVEGFantibodies or antigen binding fragment(s) thereof. One example of a VEGFantagonist is NeXstar.

Examples of categories of chemotherapeutic agents that may be used as anadditional active ingredient include but are not limited to DNA damagingagents and these include topoisomerase inhibitors (e.g., etoposide,ramptothecin, topotecan, teniposide, mitoxantrone), anti-microtubuleagents (e.g., vincristine; vinblastine), anti-metabolic agents (e.g.,cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine,6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin,chlorodeoxyadenosine), DNA alkylating agents (e.g., cisplatin,mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil,busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine,procarbazine), and DNA strand break inducing agents (e.g., bleomycin,doxorubicin, daunorubicin, idarubicin, mitomycin C). Chemotherapeuticagents include synthetic, semisynethetic and naturally derived agents.Important chemotherapeutic agents include but are not limited toAcivicin, Aclarubicin, Acodazole Hydrochloride, Acronine, Adozelesin,Adriamycin, Aldesleukin, Alitretinoin, Allopurinol Sodium, Altretamine,Ambomycin, Ametantrone Acetate, Aminoglutethimide, Amsacrine,Anastrozole, Annonaceous Acetogenins, Anthramycin, Asimicin,Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat,Benzodepa, Bexarotene, Bicalutamide, Bisantrene Hydrochloride, BisnafideDimesylate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium, Bropirimine,Bullatacin, Busulfan, Cabergoline, Cactinomycin, Calusterone,Caracemide, Carbetimer, Carboplatin, Carmustine, CarubicinHydrochloride, Carzelesin, Cedefingol, Celecoxib, Chlorambucil,Cirolemycin, Cisplatin, Cladribine, Crisnatol Mesylate,Cyclophosphamide, Cytarabine, Dacarbazine, DACA(N-[2-Dimethyl-amino)ethyl]acridine-4-carboxamide), Dactinomycin,Daunorubicin Hydrochloride, Daunomycin, Decitabine, Denileukin Diftitox,Dexormaplatin, Dezaguanine, Dezaguanine Mesylate, Diaziquone, Docetaxel,Doxorubicin, Doxorubicin Hydrochloride, Droloxifene, DroloxifeneCitrate, Dromostanolone Propionate, Duazomycin, Edatrexate, EflornithineHydrochloride, Elsamitrucin, Enloplatin, Enpromate, Epipropidine,Epirubicin Hydrochloride, Erbulozole, Esorubicin Hydrochloride,Estramustine, Estramustine Phosphate Sodium, Etanidazole, Ethiodized Oil1131, Etoposide, Etoposide Phosphate, Etoprine, Fadrozole Hydrochloride,Fazarabine, Fenretinide, Floxuridine, Fludarabine Phosphate,Fluorouracil, 5-FdUMP, Fluorocitabine, Fosquidone, Fostriecin Sodium,FK-317, FK-973, FR-66979, FR-900482, Gemcitabine, GemcitabineHydrochloride, Gemtuzumab Ozogamicin, Gold Au 198, Goserelin Acetate,Guanacone, Hydroxyurea, Idarubicin Hydrochloride, Ifosfamide,Ilmofosine, Interferon Alfa-2a, Interferon Alfa-2b, Interferon Alfa-n1,Interferon Alfa-n3, Interferon Beta-I a, Interferon Gamma-I b,Iproplatin, Irinotecan Hydrochloride, Lanreotide Acetate, Letrozole,Leuprolide Acetate, Liarozole Hydrochloride, Lometrexol Sodium,Lomustine, Losoxantrone Hydrochloride, Masoprocol, Maytansine,Mechlorethamine Hydrochloride, Megestrol Acetate, Melengestrol Acetate,Melphalan, Menogaril, Mercaptopurine, Methotrexate, Methotrexate Sodium,Methoxsalen, Metoprine, Meturedepa, Mitindomide, Mitocarcin, Mitocromin,Mitogillin, Mitomalcin, Mitomycin, Mytomycin C, Mitosper, Mitotane,Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole, Nogalamycin,Oprelvekin, Ormaplatin, Oxisuran, Paclitaxel, Pamidronate Disodium,Pegaspargase, Peliomycin, Pentamustine, Peplomycin Sulfate,Perfosfamide, Pipobroman, Piposulfan, Piroxantrone Hydrochloride,Plicamycin, Plomestane, Porfimer Sodium, Porfiromycin, Prednimustine,Procarbazine Hydrochloride, Puromycin, Puromycin Hydrochloride,Pyrazofurin, Riboprine, Rituximab, Rogletimide, Rolliniastatin,Safingol, Safingol Hydrochloride, Samarium/Lexidronam, Semustine,Simtrazene, Sparfosate Sodium, Sparsomycin, SpirogermaniumHydrochloride, Spiromustine, Spiroplatin, Squamocin, Squamotacin,Streptonigrin, Streptozocin, Strontium Chloride Sr 89, Sulofenur,Talisomycin, Taxane, Taxoid, Tecogalan Sodium, Tegafur, TeloxantroneHydrochloride, Temoporfin, Teniposide, Teroxirone, Testolactone,Thiamiprine, Thioguanine, Thiotepa, Thymitaq, Tiazofurin, Tirapazamine,Tomudex, TOP-53, Topotecan Hydrochloride, Toremifene Citrate,Trastuzumab, Trestolone Acetate, Triciribine Phosphate, Trimetrexate,Trimetrexate Glucuronate, Triptorelin, Tubulozole Hydrochloride, UracilMustard, Uredepa, Valrubicin, Vapreotide, Verteporfin, Vinblastine,Vinblastine Sulfate, Vincristine, Vincristine Sulfate, Vindesine,Vindesine Sulfate, Vinepidine Sulfate, Vinglycinate Sulfate,Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine Sulfate,Vinzolidine Sulfate, Vorozole, Zeniplatin, Zinostatin, ZorubicinHydrochloride, 2-Chlorodeoxyadenosine, 2′-Deoxyformycin,9-aminocamptothecin, raltitrexed, N-propargyl-5,8-dideazafolic acid,2-chloro-2′-arabino-fluoro-2′-deoxyadenosine,2-chloro-2′-deoxyadenosine, anisomycin, trichostatin A, hPRL-G129R,CEP-751, linomide, sulfur mustard, nitrogen mustard (mechlor ethamine),cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan,N-methyl-N-nitrosourea (MNU), N,N′-Bis(2-chloroethyl)-N-nitrosourea(BCNU), N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea (CCNU),N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU),N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nitrosourea(fotemustine), streptozotocin, diacarbazine (DTIC), mitozolomide,temozolomide, thiotepa, mitomycin C, AZQ, adozelesin, Cisplatin,Carboplatin, Ormaplatin, Oxaliplatin, CI-973, DWA 2114R, JM21.6, JM335,Bis (platinum), tomudex, azacitidine, cytarabine, gemcitabine,6-Mercaptopurine, 6-Thioguanine, Hypoxanthine, teniposide, 9-aminocamptothecin, Topotecan, CPT-11, Doxorubicin, Daunomycin, Epirubicin,darubicin, mitoxantrone, losoxantrone, Dactinomycin (Actinomycin D),amsacrine, pyrazoloacridine, all-trans retinol,14-hydroxy-retro-retinol, all-trans retinoic acid,N-(4-Hydroxyphenyl)retinamide, 13-cis retinoic acid, 3-Methyl TTNEB,9-cis retinoic acid, fludarabine (2-F-ara-AMP), and2-chlorodeoxyadenosine (2-Cda).

Other chemotherapeutic agents include: 20-epi-1,25 dihydroxyvitamin D3,5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol,adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine,amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine,anagrelide, anastrozole, andrographolide, angiogenesis inhibitors,antagonist D, antagonist G, antarelix, anti-dorsalizing morphogeneticprotein-1, antiandrogen, prostatic carcinoma, antiestrogen,antineoplaston, antisense oligonucleotides, aphidicolin glycinate,apoptosis gene modulators, apoptosis regulators, apurinic acid,ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane,atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron,azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat,BCR/ABL antagonists, benzochlorins, benzoylstaurosporine, beta lactamderivatives, beta-alethine, betaclamycin B, betulinic acid, bFGFinhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide,bistratene A, bizelesin, breflate, bleomycin A₂, bleomycin B₂,bropirimine, budotitane, buthionine sulfoximine, calcipotriol,calphostin C, camptothecin derivatives (e.g., 10-hydroxy-catnptothecin),canarypox IL-2; capecitabine, carboxamide-amino-triazole,carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor,carzelesin, casein kinase inhibitors (ICOS), castanospermine, cecropinB, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost,cis-porphyrin, cladribine, clomifene analogues, clotrimazole,collismycin A, collismycin B, combretastatin A4, combretastatinanalogue, conagenin, crambescidin 816, crisnatol, cryptophycin 8,cryptophycin A derivatives, curacin A, cyclopentanthraquinones,cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor,cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2′deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane,dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine,dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenyl spiromustine,discodermolide, docosanol, dolasetron, doxifluridine, droloxifene,dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine,edrecolomab, eflornithine, elemene, emitefur, epirubicin, epothilones(A, R═H, B, R=Me), epithilones, epristeride, estramustine analogue,estrogen agonists, estrogen antagonists, etanidazole, etoposide,etoposide 4′-phosphate (etopofos), exemestane, fadrozole, fazarabine,fenretinide, filgrastim, finasteride, flavopiridol, flezelastine,fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex,formestane, fostriecin, fotemustine, gadolinium texaphyrin, galliumnitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine,glutathione inhibitors, hepsulfam, heregulin, hexamethylenebisacetamide, homoharringtonine (HHT), hypericin, ibandronic acididarubicin, idoxifene, idramantone, ilmofosine, ilomastat,imidazoacridones, imiquimod, immunostimulant peptides, insulin-likegrowth factor-1 receptor inhibitor, interferon agonists, interferons,interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, irinotecan,iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,leukemia inhibiting factor, leukocyte alpha interferon,leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole,linear polyamine analogue, lipophilic disaccharide peptide, lipophilicplatinum compounds, lissoclinamide 7, lobaplatin, lombricine,lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine,lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides,maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysininhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone,meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone,miltefosine, mirimostim, mismatched double stranded RNA, mithracin,mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxinfibroblast growth factor-saporin, mitoxantrone, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, myriaporone, N-acetyldinaline, N-substituted benzamides,nafarelin, nagrestip, naloxone+pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, O6-benzylguanine, octreotide, okicenone,oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oralcytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin,paclitaxel analogues, paclitaxel derivatives, palauamine,palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin,pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium,pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol,phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetinB, plasminogen activator inhibitor, platinum complex, platinumcompounds, platinum-triamine complex, podophyllotoxin, porfimer sodium,porfiromycin, propyl bis-acridone, prostaglandin J2, proteasomeinhibitors, protein A-based immune modulator, protein kinase Cinhibitor, protein kinase C inhibitors, microalgal, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,purpurins, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethyleneconjugate, raf antagonists, raltitrexed, ramosetron, ras farnesylprotein transferase inhibitors, ras inhibitors, ras-GAP inhibitor,retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin,ribozymes, R11 retinamide, rogletimide, rohitukine, romurtide,roquinimex, rubiginone B1, ruboxyl, safingol, saintopin, SarCNU,sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescencederived inhibitor 1, sense oligonucleotides, signal transductioninhibitors, signal transduction modulators, single chain antigen bindingprotein, sizofuran, sobuzoxane, sodium borocaptate, sodiumphenylacetate, solverol, somatomedin binding protein, sonermin,sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin1, squalamine, stem cell inhibitor, stem-cell division inhibitors,stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glyobsaminoglycans, tallimustine, tamoxifen methiodide,tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium,telomerase inhibitors, temoporfin, temozolomide, teniposide,tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide,thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin,thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone,tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan,topsentin, toremifene, totipotent stem cell factor, translationinhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate,triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors,tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growthinhibitory factor, urokinase receptor antagonists, vapreotide, variolinB, vector system, erythrocyte gene therapy, velaresol, veramine,verdins, verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole,zanoterone, zeniplatin, zilascorb, and zinostatin stimalamer.

Other chemotherapeutic agents include: Antiproliferative agents (e.g.,Piritrexim Isothionate), Antiprostatic hypertrophy agent (e.g.,Sitogluside), Benign prostatic hyperplasia therapy agents (e.g.,Tamsulosin Hydrochloride), Prostate growth inhibitor agents (e.g.,Pentomone), and Radioactive agents: Fibrinogen 1 125, Fludeoxyglucose F18, Fluorodopa F 18, Insulin I 125, Insulin I 131, Iobenguane I 123,Iodipamide Sodium I 131, Iodoantipyrine I 131, Iodocholesterol I 131,Iodohippurate Sodium I 123, Iodohippurate Sodium I 125, IodohippurateSodium I 131, Iodopyracet I 125, Iodopyracet I 131, IofetamineHydrochloride I 123, Iomethin I 125, Iomethin I 131, Iothalamate SodiumI 125, Iothalamate Sodium I 131, Iotyrosine I 131, Liothyronine I 125,Liothyronine I 131, Merisoprol Acetate Hg 197, Merisoprol Acetate Hg203, Merisoprol Hg 197, Methyl Iodobenzo Guanine (MIBG-I131 orMIBG-I123), Selenomethionine Se 75, Technetium Tc 99m AntimonyTrisulfide Colloid, Technetium Tc 99m Bicisate, Technetium Tc 99mDisofenin, Technetium Tc 99m Etidronate, Technetium Tc 99m Exametazime,Technetium Tc 99m Furifosmin, Technetium Tc 99m Gluceptate, TechnetiumTC 99m Lidofenin, Technetium Tc 99m Mebrofenin, Technetium Tc 99mMedronate, Technetium Tc 99m Medronate Disodium, Technetium Tc 99mMertiatide, Technetium Tc 99m Oxidronate, Technetium Tc 99m Pentetate,Technetium Tc 99m Pentetate Calcium Trisodium, Technetium Tc 99mSestamibi, Technetium Tc 99m Siboroxime, Technetium Tc 99m Succimer,Technetium Tc 99m Sulfur Colloid, Technetium Tc 99m Teboroxime,Technetium Tc 99m Tetrofosmin, Technetium Tc 99m Tiatide, Thyroxine I125, Thyroxine I 131, Tolpovidone I 131, Triolein I 125, and Triolein I131. MIBG-I131 and MIBG-I123 are especially preferred chemotherapeuticagents for co-administration with the nitrofuran containingpharmaceutical compositions of the invention.

Another category of chemotherapeutic agents is anti-cancer SupplementaryPotentiating Agents, including: Tricyclic anti-depressant drugs (e.g.,imipramine, desipramine, amitryptyline, clomipramine, trimipramine,doxepin, nortriptyline, protriptyline, amoxapine and maprotiline),non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone andcitalopram), Ca⁺⁺ antagonists (e.g., verapamil, nifedipine, nitrendipineand caroverine), Calmodulin inhibitors (e.g., prenylamine,trifluoroperazine and clomipramine), Amphotericin B, Triparanolanalogues (e.g., tamoxifen), antiarrhythmic drugs (e.g., quinidine),antihypertensive drugs (e.g., reserpine), Thiol depleters (e.g.,buthionine and sulfoximine) and Multiple Drug Resistance reducing agentssuch as Cremaphor EL.

Other chemotherapeutic agents include: annonaceous acetogenins,asimicin, rolliniastatin, guanacone, squamocin, bullatacin, squamotacin,taxanes, paclitaxel, gemcitabine, methotrexate FR-900482, FK-973,FR-66979, FK-317, 5-FU, FUDR, FdUMP, Hydroxyurea, Docetaxel,discodermolide, epothilones, vincristine, vinblastine, vinorelbine,meta-pac, irinotecan, SN-38, 10-OH campto, topotecan, etoposide,adriamycin, flavopiridol, Cis-Pt, carbo-Pt, bleomycin, mitomycin C,mithramycin, capecitabine, cytarabine, 2-Cl-2′ deoxyadenosine,Fludarabine-PO₄, mitoxantrone, mitozolomide, Pentostatin, and Tomudex.

One important class of chemotherapeutic agents are taxanes (e.g.,paclitaxel and docetaxel). Nitrofuran compounds in combination withtamoxifen or aromatase inhibitor arimidex (i.e., anastrozole) areparticularly useful for breast and gynecological cancers.

Examples antibodies that can be used as other active ingredientsaccording to the invention include but are not limited to anti-CD20 mAb(monoclonal antibody), rituximab, Rituxan™, anti-CD20 mAb, tositumomabBexxar, anti-HER2, trastuzumab, Herceptin™, anti-HER2, MDX-210,anti-CA125 mAb, oregovomab, B43.13, Ovarex™, Breva-Rex, AR54, GivaRex,ProstaRex, anti-EGF receptor mAb, IMC-C225, Erbitux™, anti-EGF receptormAb, MDX-447, gemtuzumab ozogamicin, Mylotarg, CMA-676, anti-CD33 (WyethPharmaceuticals), anti-tissue factor protein (TF), (Sunol), ior-c5, c5,anti-EGF receptor mAb, MDX-447, anti-17-1A mAb, edrecolomab, Panorex,anti-CD20 mAb (Y-90 labeled), ibritumomab tiuxetan (IDEC-Y2B8), Zevalin,anti-idiotypic mAb mimic of ganglioside GD3 epitope, BEC2, anti-HLA-Dr10mAb (131 I LYM-1), Oncolym™, anti-CD33 humanized mAb (SMART M195),Zamyl™, anti-CD52 humAb (LDP-03), CAMPATH, anti-CD1 mAb, for t6,anti-CAR (complement activating receptor) mAb, MDX-11, humanizedbispecific mAb conjugates (complement cascade activators), MDX-22, OV103(Y-90 labeled antibody), celogovab, OncoScint™, anti-17-1A mAb, 3622W94,anti-VEGF (RhumAb-VEGF), bevacizumab, Avastin™, anti-TAC (IL-2 receptor)humanized Ab (SMART), daclizumab, Zenapax, anti-TAG-72 partiallyhumanized bispecific Ab, MDX-220, anti-idiotypic mAb mimic of highmolecular weight proteoglycan (I-Mel-I), MELIMMUNE-1, anti-idiotypic mAbmimic of high molecular weight proteoglycan (I-MeI-2), MELIMMUNE-2,anti-CEA Ab (hMN14), CEACide™, Pretarget™ radioactive targeting agents,hmAbH11 scFv fragment (NovomAb-G2), H11 scFv, anti-DNA or DNA-associatedproteins (histones) mAb and conjugates, TNT (e.g. Cotara™), Gliomab-HmAb, GNI-250 mAb, anti-EGF receptor mAb, EMD-72000, anti-CD22 humanizedAb, LymphoCide, Non-Hodgkin's anti-CD33 mAb conjugate with calicheamicin(CMA 676), gemtuzumab ozogamicin, Mylotarg™, Monopharm-C, colon,anti-idiotypic human mAb to GD2 ganglioside, 4B5, melanoma, anti-EGFreceptor humanized Ab, ior egf/r3, anti-ior c2 glycoprotein mAb, for c5,BABS (biosynthetic antibody binding site) proteins, anti-FLK-2/FLT-3mAb, mAb/small-molecule conjugate, TAP (tumor-activated prodrug),anti-GD-2 bispecific mAb, MDX-260, antinuclear autoantibodies (bindsnucleosomes), ANA Ab, anti-HLA-DR Ab (SMART 1D10 Ab), Remitogen™, SMARTABL 364 Ab, anti-CEA 1131-labeled mAb, ImmuRAIT-CEA.

Other antibodies that can be used according to the invention includeanti-TNFα antibody such as infliximab (Remicade) and etanercept (Enbrel)for rheumatoid arthritis and Crohn's disease, palivizuma, anti-RSVantibody for pediatric subjects, bevacizumab, alemtuzumab, Campath-1H,BLyS-mAb, fSLE; anti-VEGF2, anti-Trail receptor; B3 mAb, m170 mAb, mABBR96, and Abx-Cbl mAb. The invention embraces a number of classes ofantibodies and fragments thereof including but not limited to antibodiesdirected to cancer antigens (as described above), cell surface molecule,stromal cell molecules, extracellular matrix molecules, and tumorvasculature associated molecules.

A cell surface molecule is a molecule that is expressed at the surfaceof a cell. In addition to an extracellular domain, it may furthercomprise a transmembrane domain and a cytoplasmic domain. Examplesinclude HER 2, CD20, CD33, EGF receptor, HLA markers such as HLA-DR,CD52, CD1, CEA, CD22, GD2 ganglioside, FLK2/FLT3, VEGF, VEGFR, and thelike.

A stromal cell molecule is a molecule expressed by a stromal cell.Examples include but are not limited to FAP and CD26.

An extracellular matrix molecule is a molecule found in theextracellular matrix. Examples include but are not limited to collagen,glycosaminoglycans (GAGs), proteoglycans, elastin, fibronectin andlaminin.

A tumor vasculature associated molecule is a molecule expressed byvasculature of a tumor (i.e., a solid cancer rather than a systemiccancer such as leukemia). As with a cancer antigen, a tumor vasculatureassociated molecule may be expressed by normal vasculature however itspresence on vasculature of a tumor makes it a suitable target foranti-cancer therapy. In some instances, the tumor vasculature associatedmolecule is expressed at a higher level in tumor vasculature than it isin normal vasculature. Examples include but are not limited to endoglin(see U.S. Pat. No. 5,660,827), ELAM-1, VCAM-1, ICAM-1, ligand reactivewith LAM-1, MHC class II antigens, aminophospholipids such asphosphatidylserine and phosphatidylethanolamine (as described in U.S.Pat. No. 6,312,694), VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1), and othertumor vasculature associated antigens such as those described in U.S.Pat. No. 5,776,427. Antibodies to endoglin are described in U.S. Pat.No. 5,660,827 and include TEC-4 and TEC-11, and antibodies thatrecognize identical epitopes to these antibodies. Antibodies toaminophospholipids are described in U.S. Pat. No. 6,312,694. Antibodiesthat inhibit VEGF are described in U.S. Pat. No. 6,342,219 and include2C3 (ATCC PTA 1595). Other antibodies that are specific for tumorvasculature include antibodies that react to a complex of a growthfactor and its receptor such as a complex of FGF and the FGFR or acomplex of TGFβ and the TGFβR. Antibodies of this latter class aredescribed in U.S. Pat. No. 5,965,132, and include GV39 and GV97.

It is to be understood that the antibodies embraced by the inventioninclude those recited explicitly herein and also those that bind to thesame epitope as those recited herein.

Also useful in the invention are antibodies such as the following, allof which are commercially available:

Apoptosis Antibodies:

BAX Antibodies: Anti-Human Bax Antibodies (Monoclonal), Anti-Human BaxAntibodies (Polyclonal), Anti-Murine Bax Antibodies (Monoclonal),Anti-Murine Bax Antibodies (Polyclonal);

Fas/Fas Ligand Antibodies: Anti-Human Fas/Fas Ligand Antibodies,Anti-Murine Fas/Fas Ligand Antibodies Granzyme Antibodies Granzyme BAntibodies;

BCL Antibodies: Anti Cytochrome C Antibodies, Anti-Human BCL Antibodies(Monoclonal), Anti-Human bcl Antibodies (Polyclonal), Anti-Murine bclAntibodie, (Monoclonal), Anti-Murine bcl Antibodies (Polyclonal)

Miscellaneous Apoptosis Antibodies: Anti TRADD, TRAIL, TRAFF, DR3Antibodies Anti-Human Fas/Fas Ligand Antibodies Anti-Murine Fas/FasLigand Antibodies;

Miscellaneous Apoptosis Related Antibodies: BIM Antibodies: Anti Human,Murine bim Antibodies (Polyclonal), Anti-Human, Murine bim Antibodies(Monoclonal);

PARP Antibodies: Anti-Human PARP Antibodies (Monoclonal), Anti-HumanPARP Antibodies (Polyclonal) Anti-Murine PARP Antibodies;

Caspase Antibodies: Anti-Human Caspase Antibodies (Monoclonal),Anti-Murine Caspase Antibodies;

Anti-CD Antibodies: Anti-CD29, PL18-5 PanVera, Anti-CD29, PL4-3 PanVera,Anti-CD41a, PT25-2 PanVera, Anti-CD42b, PL52-4 PanVera, Anti-CD42b,GUR20-5 PanVera, Anti-CD42b, WGA-3 PanVeraAnti-CD43, 1D4 PanVera,Anti-CD46, MCP75-6 PanVera, Anti-CD61, PL11-7 PanVera, Anti-CD61, PL8-5PanVera, Anti-CD62/P-slctn, PL7-6 PanVera, Anti-CD62/P-slctn, WGA-1PanVera, Anti-CD154, 5F3 PanVera; and anti-CD1, anti-CD2, anti-CD3,anti-CD4, anti-CD5, anti-CD6, anti-CD7, anti-CD8, anti-CD9, anti-CD10,anti-CD11, anti-CD12, anti-CD13, anti-CD14, anti-CD15, anti-CD16,anti-CD17, anti-CD18, anti-CD19, anti-CD20, anti-CD21, anti-CD22,anti-CD23, anti-CD24, anti-CD25, anti-CD26, anti-CD27, anti-CD28,anti-CD29, anti-CD30, anti-CD31, anti-CD32, anti-CD33, anti-CD34,anti-CD35, anti-CD36, anti-CD37, anti-CD38, anti-CD39, anti-CD40anti-CD41, anti-CD42, anti-CD43, anti-CD44, anti-CD45, anti-CD46,anti-CD47, anti-CD48, anti-CD49, anti-CD50, anti-CD51, anti-CD52,anti-CD53, anti-CD54, anti-CD55, anti-CD56, anti-CD57, anti-CD58,anti-CD59, anti-CD60, anti-CD61, anti-CD62, anti-CD63, anti-CD64,anti-CD65, anti-CD66, anti-CD67, anti-CD68, anti-CD69, anti-CD70,anti-CD71, anti-CD72, anti-CD73, anti-CD74, anti-CD75, anti-CD76,anti-CD77, anti-CD78, anti-CD79, anti-CD80, anti-CD81, anti-CD82,anti-CD83, anti-CD84, anti-CD85, anti-CD86, anti-CD87, anti-CD88,anti-CD89, anti-CD90, anti-CD91, anti-CD92, anti-CD93, anti-CD94,anti-CD95, anti-CD96, anti-CD97, anti-CD98, anti-CD99, anti-CD100,anti-CD101, anti-CD102, anti-CD103, anti-CD104, anti-CD105, anti-CD106,anti-CD107, anti-CD108, anti-CD109, anti-CD110, anti-CD111, anti-CD112,anti-CD113, anti-CD114, anti-CD115, anti-CD116, anti-CD117, anti-CD118,anti-CD119, anti-CD120, anti-CD121, anti-CD122, anti-CD123, anti-CD124,anti-CD125, anti-CD126, anti-CD127, anti-CD128, anti-CD129, anti-CD130,anti-CD131, anti-CD132, anti-CD133, anti-CD134, anti-CD135, anti-CD136,anti-CD137, anti-CD138, anti-CD139, anti-CD140, anti-CD141, anti-CD142,anti-CD143, anti-CD144, anti-CD145, anti-CD146, anti-CD147, anti-CD148,anti-CD149, anti-CD150, anti-CD151, anti-CD152, anti-CD153, anti-CD154,anti-CD155, anti-CD156, anti-CD157, anti-CD158, anti-CD159, anti-CD160,anti-CD161, anti-CD162, anti-CD163, anti-CD164, anti-CD165, anti-CD166,anti-CD167, anti-CD168, anti-CD169, anti-CD170, anti-CD171, anti-CD172,anti-CD173, anti-CD174, anti-CD175, anti-CD176, anti-CD177, anti-CD178,anti-CD179, anti-CD180, anti-CD181, anti-CD182, anti-CD183, anti-CD184,anti-CD185, anti-CD186, anti-CD187, anti-CD188, anti-CD189, anti-CD190,anti-CD191, anti-CD192, anti-CD193, anti-CD194, anti-CD195, anti-CD196,anti-CD197, anti-CD198, anti-CD199, anti-CD200, anti-CD201, anti-CD202,anti-CD203, anti-CD204, anti-CD265, anti-CD206, anti-CD207, anti-CD208,anti-CD209, anti-CD210, anti-CD211, anti-CD212, anti-CD213, anti-CD214,anti-CD215, anti-CD216, anti-CD217, anti-CD218, anti-CD219, anti-CD220,anti-CD221, anti-CD222, anti-CD223, anti-CD224, anti-CD225, anti-CD226,anti-CD227, anti-CD228, anti-CD229, anti-CD230, anti-CD231, anti-CD232,anti-CD233, anti-CD234, anti-CD235, anti-CD236, anti-CD237, anti-CD238,anti-CD239, anti-CD240 anti-CD241, anti-CD242, anti-CD243, anti-CD244,anti-CD245, anti-CD246, anti-CD247, anti-CD248, anti-CD249, anti-CD250,and the like.

Human Chemokine Antibodies: Human CNTF Antibodies, Human EotaxinAntibodies, Human Epitherlial Neutrophil Activating Peptide-78, HumanExodus Antibodies, Human GRO Antibodies, Human HCC-1 Antibodies, Human1-309 Antibodies, Human IP-10 Antibodies, Human I-TAC Antibodies, HumanLIF Antibodies, Human Liver-Expressed Chemokine Antibodies, Humanlymphotoxin Antibodies, Human MCP Antibodies, Human MIP Antibodies,Human Monokine Induced by IFN-gamma Antibodies, Human NAP-2 Antibodies,Human NP-1 Antibodies, Human Platelet Factor-4 Antibodies, Human RANTESAntibodies, Human SDF Antibodies, Human TECK Antibodies;

Murine Chemokine Antibodies: Human B-Cell Attracting Murine ChemokineAntibodies, Chemokine-1 Antibodies, Murine Eotaxin Antibodies, MurineExodus Antibodies, Murine GCP-2 Antibodies, Murine KC Antibodies, MurineMCP Antibodies, Murine MIP Antibodies, Murine RANTES Antibodies, RatChemokine Antibodies, Rat Chemokine Antibodies, Rat CNTF Antibodies, RatGRO Antibodies, Rat MCP Antibodies, Rat MIP Antibodies, Rat RANTESAntibodies;

Cytokine/Cytokine Receptor Antibodies: Human BiotinylatedCytokine/Cytokine Receptor Antibodies, Human IFN Antibodies, Human ILAntibodies, Human Leptin Antibodies, Human Oncostatin Antibodies, HumanTNF Antibodies, Human TNF Receptor Family Antibodies, MurineBiotinylated Cytokine/Cytokine Receptor Antibodies, Murine IFNAntibodies, Murine IL Antibodies, Murine TNF Antibodies, Murine TNFReceptor Antibodies; anti-CCR4 antibody;

Rat Cytokine/Cytokine Receptor Antibodies: Rat BiotinylatedCytokine/Cytokine Receptor Antibodies, Rat IFN Antibodies, Rat ILAntibodies, Rat TNF Antibodies;

ECM Antibodies: Collagen/Procollagen, Laminin, Collagen (Human), Laminin(Human), Procollagen (Human), Vitronectin/Vitronectin Receptor,Vitronectin (Human), Vitronectin Receptor (Human),Fibronectin/Fibronectin Receptor, Fibronectin (Human), FibronectinReceptor (Human);

Growth Factor Antibodies: Human Growth Factor Antibodies, Murine GrowthFactor Antibodies, Porcine Growth Factor Antibodies;

Miscellaneous Antibodies: B aculovirus Antibodies, Cadherin Antibodies,Complement Antibodies, Clq Antibodies, VonWillebrand Factor Antibodies,Cre Antibodies, HIV Antibodies, Influenza Antibodies, Human Leptin.Antibodies, Murine Leptin Antibodies, Murine CTLA-4 Antibodies, HumanCTLA-4 Antibodies, P450 Antibodies, RNA Polymerase Antibodies;

Neurobio Antibodies: Amyloid Antibodies, GFAP Antibodies, Human NGFAntibodies, Human NT-3 Antibodies, Human NT-4 Antibodies:

Still other antibodies can be used in the invention and these includeantibodies listed in references such as the MSRS Catalog of PrimaryAntibodies, and Linscott's Directory.

In some preferred embodiments of the invention, the antibodies areAvastin (bevacizumab), BEC2 (mitumomab), Bexxar (tositumomab), Campath(alemtuzumab), CeaVac, Herceptin (trastuzumab), IMC-C225 (centuxitmab),LymphoCide (epratuzumab), MDX-210, Mylotarg (gemtuzumab ozogamicin),Panorex (edrecolomab), Rituxan (rituximab), Theragyn (pemtumomab),Zamyl, and Zevalin (ibritumomab tituxetan). The invention also coversantibody fragments thereof.

In some preferred embodiments, the cancer antigen is VEGF,Anti-idiotypic mAb (GD3 ganglioside mimic), CD20, CD52; Anti-idiotypicmAb (CEA mimic), ERBB2, EGFR, CD22, ERBB2 X CD65 (fcyRl), EpCam, PEM andCD33.

The invention encompasses the use of both antibodies and antibodyfragments. The antibodies may be monoclonal or polyclonal, and can beprepared by conventional methodology. They may further be isolated orpresent in an ascites fluid. Such antibodies can be further manipulatedto create chimeric or humanized antibodies as will be discussed ingreater detail below.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′, region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of co-specificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions hasbeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies. Commercial sources of humanized orchimeric antibodies include GenPharm, Xenotech, AbGenix and CellGeneSys.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

The nitrofuran compounds are administered in therapeutically effectiveand physiologically acceptable amounts, which is the amountphysiologically tolerable to the subject that is necessary or sufficientto realize the desired beneficial biologic effect, in this case thetreatment of cancer or the inhibition of angiogenesis. A biologicallybeneficial effect can, for example, be measured by determining thephysiological effects of the treatment following administration of thetreatment. The biologically beneficial effect may be the ameliorationand or absolute elimination of symptoms resulting from the disorderbeing treated, or the inhibition of angiogenesis in the disorder beingtreated as evidenced, for example, by a reduction in the number ofmicrovessels (e.g., abnormal microvessels) on imaging.

The therapeutically effective and physiologically acceptable amount mayvary depending upon the particular compound(s) or combination ofcompounds and/or therapies used. It can also vary depending on suchfactors as the condition (e.g. cancer) being treated, the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art can empirically determine the effective amount of aparticular nitrofuran compound or combination without necessitatingundue experimentation. Combined with the teachings provided herein, bychoosing among the various compounds and weighing factors such aspotency, relative bioavailability, patient body weight, severity ofadverse side-effects and preferred mode of administration, an effectiveprophylactic or therapeutic treatment regimen can be planned which doesnot cause substantial toxicity and yet is entirely effective to treatthe particular subject.

In some instances, a sub-therapeutic dosage of either the nitrofurancompound or the second agent, or a sub-therapeutic dosage of both, isused to treat the subject. For example, when a nitrofuran compound isused together with an anti-cancer agent, the nitrofuran compound and theanti-cancer agent may be administered in sub-therapeutic doses and stillproduce a desirable therapeutic effect. A “sub-therapeutic dose” as usedherein refers to a dosage which is less than that dosage which wouldproduce a therapeutic result in the subject if administered in theabsence of the other agent. Thus, the sub-therapeutic dose of ananti-cancer agent is one which would not produce the same or asubstantially similar therapeutic result in the subject in the absenceof the administration of nitrofuran compound. Therapeutic doses ofanti-cancer agents are known in the field of medicine. These doses havebeen extensively described in references such as Remington'sPharmaceutical Sciences, 18th ed., 1990, or the Physician DesktopReference; as well as many other medical references relied upon by themedical profession as guidance for the treatment of cancer and are wellknown in the art.

For any compound described herein a therapeutically effective amount maybe initially determined from in vitro assays such as cell cultureassays. Therapeutically effective amounts can also be determined inanimal studies. For instance, the effective amount of a nitrofurancompound with or without a second agent can be assessed using in vivoassays of, for example, tumor regression and/or prevention of tumorformation. Relevant animal models include, for example, assays in whichmalignant cells are injected into the animal subjects, usually in adefined site. Generally, a range of nitrofuran compound doses areadministered into the animal. Inhibition of the growth of a tumorfollowing the injection of the malignant cells is indicative of theability to reduce the risk of developing a cancer. Inhibition of furthergrowth (or reduction in size) of a pre-existing tumor is indicative ofthe ability to treat the cancer.

The applied dose of both agents can be adjusted based on the relativebioavailability and potency of the administered compound(s). Adjustingthe dose to achieve maximal efficacy based on the methods describedabove and other methods are well within the capabilities of theordinarily skilled artisan.

Subject doses of the compounds described herein typically range fromabout 0.1 μg to 30,000 mg, more typically from about 1 μg/day to 20,000mg, even more typically from about 10 μg to 15,000 mg, and mosttypically from about 100 μg to 10,000 Stated in terms of subject bodyweight, typical dosages range from about 0.1 μg to 200 mg/kg/day, moretypically from about 0.5 to 150 mg/kg/day. In some importantembodiments, the compound is administered in amounts from about 1 to 100mg/kg/day. In some other important embodiments, the compound isadministered in an amount of 10-60 mg/kg/day.

A “routine schedule” as used herein, refers to a predetermineddesignated period of time. The routine schedule may encompass periods oftime which are identical or which differ in length, as long as theschedule is predetermined. For instance, the routine schedule mayinvolve 2, 3, 4, or 6 administrations per day, administration on a dailybasis, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between, every two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months, etc. Alternatively,the predetermined routine schedule may involve administration on a dailybasis for the first week, followed by a monthly basis for severalmonths, and then every three months after that. Any particularcombination would be covered by the routine schedule as long as it isdetermined ahead of time that the appropriate schedule involvesadministration on a certain day.

The compounds of the invention may be administered in pharmaceuticallyacceptable carriers, or in the context of a vector or delivery system.An example of a chemical/physical vector of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2-4.0 μm can encapsulate largemacromolecules. RNA, DNA and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., (1981) 6:77).

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a sugar, glycolipid, or protein.Ligands which may be useful for targeting a liposome to a cell include,but are not limited to intact or fragments of molecules which interactwith cell specific receptors and molecules, such as antibodies, whichinteract with the cell surface markers of cells. Such ligands may easilybe identified by binding assays well known to those of skill in the art.In still other embodiments, the liposome may be targeted to the cancerby coupling it, for example, to one of the immunotherapeutic antibodiesdiscussed earlier. Additionally, the vector may be coupled to a nucleartargeting peptide, which will direct the vector to the nucleus of thehost cell.

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACET™, which are formed of cationic lipids suchas N-[1-(2, 3 dioleyloxy)-propyl]-N,N, N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, (1985) 3:235-241.

In another embodiment, the chemical/physical vector is a biocompatiblemicrosphere that is suitable for delivery, such as oral or mucosaldelivery. Such microspheres are disclosed in Chickering et al., Biotech.And Bioeng., (1996) 52:96-101 and Mathiowitz et al., Nature, (1997)386:410-414 and PCT Patent Application WO97/03702.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the nitrofuran compound and/or the second agent to thesubject. Biodegradable matrices are preferred. Such polymers may benatural or synthetic polymers. The polymer is selected based on theperiod of time over which release is desired, generally in the order ofa few hours to a year or longer. Typically, release over a periodranging from between a few hours and three to twelve months is mostdesirable. The polymer optionally is in the form of a hydrogel that canabsorb up to about 90% of its weight in water and further, optionally iscross-linked with multi-valent ions or other polymers.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein the agents are dispersed throughout a solidpolymeric matrix) or a microcapsule (wherein the agents are stored inthe core of a polymeric shell). Other forms of the polymeric matrix forcontaining the agents include films, coatings, gels, implants, andstents. The size and composition of the polymeric matrix device isselected to result in favorable release kinetics in the tissue intowhich the matrix is introduced. The size of the polymeric matrix furtheris selected according to the method of delivery which is to be used,typically injection into a tissue or administration of a suspension byaerosol into the nasal and/or pulmonary areas. Preferably when anaerosol route is used the polymeric matrix and the nitrofuran compoundare encompassed in a surfactant vehicle. The polymeric matrixcomposition can be selected to have both favorable degradation rates andalso to be formed of a material which is bioadhesive, to furtherincrease the effectiveness of transfer when the matrix is administeredto a nasal and/or pulmonary surface that has sustained an injury. Thematrix composition also can be selected not to degrade, but rather, torelease by diffusion over an extended period of time. In some preferredembodiments, the nitrofuran compound is administered to the subject viaan implant.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(laurel methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

The compositions and methods of the invention in certain instances maybe useful for replacing existing surgical procedures or drug therapies,although in most instances the present invention is useful in improvingthe efficacy of existing therapies for treating such conditions.Accordingly combination therapy may be used to treat the subjects thatare undergoing or that will undergo a treatment for cancer. For example,the agents may be administered to a subject in combination with anotheranti-proliferative (e.g., an anti-cancer) therapy. Suitable anti-cancertherapies include surgical procedures to remove the tumor mass,chemotherapy or localized radiation. The other anti-proliferativetherapy may be administered before, concurrent with, or after treatmentwith the agent of the invention. There may also be a delay of severalhours, days and in some instances weeks between the administration ofthe different treatments, such that the agent may be administered beforeor after the other treatment. In some embodiments, the nitrofurancompound may be administered with or without the otheranti-proliferative treatment (e.g., prior to surgery, radiation orchemotherapy), although the timing is not so limited.

The nitrofuran compound can also be administered in combination withnon-surgical, anti-proliferative (e.g., anti-cancer) drug therapy. Inone embodiment, the agent may be administered in combination with ananti-cancer agent such as a cytostatic compound. A cytostatic compoundis a compound (e.g., a nucleic acid, a protein) that suppresses cellgrowth and/or proliferation. In some embodiments, the cytostaticcompound is directed towards the malignant cells of a tumor. In yetother embodiments, the cytostatic compound is one which inhibits thegrowth and/or proliferation of vascular smooth muscle cells orfibroblasts.

According to the methods of the invention, the nitrofuran or nitrofurananalog may be administered prior to, concurrent with, or following otheranti-cancer agent(s). The administration schedule may involveadministering the different agents in an alternating fashion. In otherembodiments, the combination therapy of the invention may be deliveredbefore and during, or during and after, or before and after treatmentwith other therapies. In some cases, the agent is administered more than24 hours before the administration of the other anti-proliferativetreatment. In other embodiments, more than one anti-proliferativetherapy may be administered to a subject. For example, the subject mayreceive the agents of the invention, in combination with both surgeryand at least one other anti-proliferative compound. Alternatively, theagent may be administered in combination with more than one anti-canceragent.

The nitrofuran compound can be combined with other therapeutic agentssuch as adjuvants to enhance immune responses. The nitrofuran ornitrofuran analog and other therapeutic agent(s) may be administeredsimultaneously or sequentially. When the other therapeutic agents areadministered simultaneously they can be administered in the same orseparate formulations, but are administered at the same time. Theadministration of the other therapeutic agents and the nitrofuran ornitrofuran analog can also be temporally separated, meaning that thetherapeutic agents are administered at a different time, either beforeor after, the administration of the nitrofuran or nitrofuran analog. Theseparation in time between the administration of these compounds andagents may be a matter of minutes or it may be longer. Other therapeuticagents include but are not limited to nucleic acid adjuvants,non-nucleic acid adjuvants, cytokines, non-immunotherapeutic antibodies,antigens, etc.

A nucleic acid adjuvant is an adjuvant that is a nucleic acid. Examplesinclude immunostimulatory nucleic acid molecules such as thosecontaining CpG dinucleotides, as described in U.S. Pat. No. 6,194,388B1, issued Feb. 27, 2001, U.S. Pat. No. 6,207,646 B1, issued Mar. 27,2001, and U.S. Pat. No. 6,239,116 B1, issued May 29, 2001.

A “non-nucleic acid adjuvant” is any molecule or compound except for theimmunostimulatory nucleic acids described herein which can stimulate thehumoral and/or cellular immune response. Non-nucleic acid adjuvantsinclude, for instance, adjuvants that create a depo effect,immune-stimulating adjuvants, adjuvants that create a depo effect andstimulate the immune system and mucosal adjuvants.

An “adjuvant that creates a depo effect” as used herein is an adjuvantthat causes an antigen, such as a cancer antigen present in a cancervaccine, to be slowly released in the body, thus prolonging the exposureof immune cells to the antigen. This class of adjuvants includes but isnot limited to alum (e.g., aluminum hydroxide, aluminum phosphate); oremulsion-based formulations including mineral oil, non-mineral oil,water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsionssuch as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsionstabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville,Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizingdetergent and a micelle-forming agent; IDEC Pharmaceuticals Corporation,San Diego, Calif.).

An “immune stimulating adjuvant” is an adjuvant that causes activationof a cell of the immune system. It may, for instance, cause an immunecell to produce and secrete cytokines. This class of adjuvants includesbut is not limited to saponins purified from the bark of the Q.saponaria tree, such as QS21 (a glycolipid that elutes in the 21^(st)peak with HPLC fractionation; Antigenics, Inc., Waltham, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

“Adjuvants that create a depo effect and stimulate, the immune system”are those compounds which have both of the above-identified functions.This class of adjuvants includes but is not limited to ISCOMS(Immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2which is an oil-in-water emulsion containing MPL and QS21: SmithKlineBeecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKlineBeecham adjuvant system #4 which contains alum and MPL; SBB, Belgium);non-ionic block copolymers that form micelles such as CRL 1005 (thesecontain a linear chain of hydrophobic polyoxpropylene flanked by chainsof polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex AdjuvantFormulation (SAF, an oil-in-water emulsion containing Tween 80 and anonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

A “non-nucleic acid mucosal adjuvant” as used herein is an adjuvantother than an immunostimulatory nucleic acid that is capable of inducinga mucosal immune response in a subject when administered to a mucosalsurface in conjunction with an antigen. Mucosal adjuvants include butare not limited to Bacterial toxins: e.g., Cholera toxin (CT), CTderivatives including but not limited to CT B subunit (CTB) (Wu et al.,1998, Tochikubo et al., 1998); CTD53 (Val to Asp) (Fontana et al.,1995); CTK97 (Val to Lys) (Fontana et al., 1995); CTK104 (Tyr to Lys)(Fontana et al., 1995); CTD53/K63 (Val to Asp, Ser to Lys) (Fontana etal., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN107 (H is toAsn) (Fontana et al., 1995); CTE114 (Ser to Glu) (Fontana et al., 1995);CTE112K (Glu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe)(Yamamoto et al., 1997a, 1997b); CTS106 (Pro to Lys) (Douce et al.,1997, Fontana et al., 1995); and CTK63 (Ser to Lys) (Douce et al., 1997,Fontana et al., 1995), Zonula occludens toxin, zot, Escherichia coliheat-labile enterotoxin, Labile Toxin (Li), LT derivatives including butnot limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg toLys) (Komase et al., 1998, Douce et al., 1995); LT61F (Ser to Phe)(Komase et al., 1998); LT112K (Glu to Lys) (Komase et al., 1998); LT118E(Gly to Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al.,1998); LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to Lys)(Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al.,1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), Pertussis toxin,PT. (Lycke et al., 1992, Spangler B D, 1992, Freytag and Clemments,1999, Roberts et al., 1995, Wilson et al.; 1995) including PT-9K/129G(Roberts et al., 1995, Cropley et al., 1995); Toxin derivatives (seebelow) (Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al.,1995, Freytag and Clements, 1999); Lipid A derivatives (e.g.,monophosphoryl lipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998;Muramyl Dipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa etal., 1989, Michalek et al., 1983, Morisaki et al., 1983); Bacterialouter membrane proteins (e.g., outer surface protein A (OspA)lipoprotein of Borrelia burgdorferi, outer membrane protein of Neisseriameningitidis) (Mannaro et al., 1999, Van de Verg et al., 1996);Oil-in-water emulsions (e.g., MF59) (Barchfield et al., 1999, Verschooret al., 1999, O'Hagan, 1998); Aluminum salts (Isaka et al., 1998, 1999);and Saponins (e.g., QS21) Aquila Biopharmaceuticals, Inc., Worcester,Mass.) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMS, MF-59 (asqualene-in-water emulsion stabilized with Span 85 and Tween 80; ChironCorporation, Emeryville, Calif.); the Seppic ISA series of Montanideadjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX(an oil-in-water emulsion containing a stabilizing detergent and amicell-forming agent; IDEC Pharmaceuticals Corporation, San Diego,Calif.); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc.,Boulder, Colo.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA) and Leishmania elongation factor (CorixaCorporation, Seattle, Wash.).

The invention further provides kits that comprise the compounds and/oragents of the invention and optionally instructions of use thereof. Thecompounds and/or agents may be present in parenteral forms (e.g., forintravenous, intramuscular, or intrathecal administration) or in oralforms such as tablets, pills, capsules, caplets and the like. The kitmay further contain a second active ingredient or agent eitherformulated together with the nitrofuran or formulated separately. Theunit dosages provided in each form will depend upon whether thenitrofuran compound is used together with or in the absence of thesecond ingredient or agent. The kit may optionally comprise a housingsuch as a box or a bag. Instructions for use may be supplied separatelyfrom the dispensing unit or housing or they may be imprinted on one orboth. The compounds and/or agents may be provided in a one a daydispensing unit such as a blister pack or dial pack type dispenser,preferably with days of the week or day of the month (e.g., 1, 2, 3, 4,etc.) printed on the dispenser. If the compounds and/or agents are to beadministered every other day or twice (or more) a day, the dispensingunit can be modified accordingly.

The following examples are provided to illustrate specific instances ofthe practice of the present invention. They are not intended to limitthe scope of the invention. As will be apparent to one of ordinary skillin the art, the present invention will find application in a variety ofmethods and compositions. In the examples, the therapeutic potential ofnifurtimox on neuroblastoma using three neuroblastoma cell lines(SMS-KCNR, SMS-KCN, and IMR-32) which are representative of the Type 3childhood form of neuroblastom¹⁶, is demonstrated. Without intending tobe bound by any particular theory, it is believed that the molecularmechanism of action of nifurtimox involves formation of free radicalswithin cells^(9,17). Cellular damage caused by free radicals result ininduction of apoptosis leading to the observed toxicity bynifurtimox¹⁸⁻²⁰. Earlier studies have shown that the treatment of T.cruzi epimastigotes with nifurtimox decreased cell viability which wasassociated with increased ultrastructural damage in the cell^(10,21). Asimilar cytotoxic effect was observed by nifurtimox on SMS-KCNR, SMS-KCNand IMR-32 neuroblastoma cell lines as described in Example 4. Among thedifferent neuroblastoma cell lines used, SMS-KCNR was found to be mostsensitive to nifurtimox and further studies were carried out using thiscell line. Nifurtimox showed a dose and time dependent decrease in cellviability (FIG. 2A). Decrease in cell viability may be due to cell deathdue to cytotoxicity of the drug or lack of proliferation. Our studiesdemonstrated dose dependent decrease in cell proliferation using BrdUassay as detailed in Example 5. The cytotoxic effect of nifurtimox wasevident during microscopic examination as described in Example 6, duringwhich, the cells were found to be rounding up and floating suggestive ofapoptosis (FIG. 3A). TUNEL assay was performed to confirm apoptosis. Inthis assay, the DNA termini generated following fragmentation, as in thecase of apoptosis, were labeled with fluorescent deoxy-thymidine analogusing the terminal deoxynucleotide transferase enzyme. Increased numberof terminal ends in the DNA leads to increased labeling which in turn isreflected in the increased fluorescence signal²². Increased TUNELstaining was observed with nifurtimox treatment (FIG. 3B) whichindicated that the drug caused apoptosis in the neuroblastoma cells.

Caspases are a central component of apoptotic machinery and caspase-3 isan executioner caspase that is activated by several anti-cancer drugs²³.As detailed in Example 9, upon treatment with nifurtimox, there wasstrong activation of caspase-3. This is shown by an immunoblot oftreated cells with antibodies specific to activated caspase-3 (FIG. 4A).Caspase-3 activation was dose dependent which corresponded withincreased apoptosis marked by increased TUNEL staining of the treatedcells.

The role of caspases in the nifurtimox induced apoptosis wascorroborated by using caspase inhibitors. Pretreatment of cells withZ-VAD-FMK, a pan-caspase inhibitor²⁴, which has the ability to inhibit abroad range of caspases effectively, reversed nifurtimox inducedapoptosis in the neuroblastoma cells (FIG. 4B). This reversal ofapoptosis by Z-VAD-FMK confirmed the involvement of caspases in thenifurtimox induced apoptosis of neuroblastoma cells. The pan-caspaseinhibitor, Z-VAD-FMK, has been used by others to confirm caspasemediated apoptosis by Flavopiridol treated neuroblastoma cell lines²⁵.

TrkB is a receptor tyrosine kinase and is a high affinity receptor forneurotrophins. Activation of TrkB by neurotropins, similar to otherTrks, has been implicated in differentiation as well as in suppressingapoptosis in neuronal cells²⁶. TrkB is expressed almost exclusively inbiologically unfavorable neuroblastomas^(2,3). BDNF binding to TrkBleads to the activation of the Ras/MAPK and PI3K/Akt signaling pathways.The Akt pathway has been shown to be critical for cell survival andresistance to chemotherapy^(3,15). As detailed in Example 3, whenSMS-KCNR cells were stimulated with BDNF, phosphorylation of Akt due toactivation of the TrkB receptor was inhibited in the presence ofnifurtimox (FIG. 6A). This suggests that nifurtimox may act byinhibiting TrkB signaling. This finding is significant because theTrkB-BDNF pathway promotes cell survival and is known to protect cellsfrom DNA damaging therapeutic agents in neuroblastoma cells leading tothe development of chemoresistance^(3,18,27). Therefore drugs thatinhibit this pathway are valuable in treating neuroblastoma.Additionally, nifurtimox decreased phosphorylation of Akt (FIG. 6B)suggesting that Akt may be a direct target of nifurtimox.

Similar to Akt, ERK1/2 phosphorylation is considered to promote survivaland is activated by BDNF/TrkB pathway in neuroblastoma²⁶. However,treatment of SMS-KCNR cells with nifurtimox did not alter thephosphorylation of ERK 1/2 phosphorylation (data not shown). Thesedifferential effects of nifurtimox likely reflect the unique functionsof each pathway. For example, it has been reported that Akt mediates thepromotion of cell survival induced by BDNF, where as, ERK1/2 mediatesBDNF induced cell differentiation²⁸. These studies indicate thatnifurtimox can be a therapeutic agent to combat neuroblastoma.

Tested in vivo, Niurtimox treatment resulted in a significant decreasein tumor size in mice. See Example 12. In humans, Nifurtimox treatment,alone or in combination with other therapies, resulted in tumorregression without toxic side effects. Other researchers have found thatNifurtimox has many side effects in adults including nausea/vomiting,myalgia, weakness, headache, parasthesias, polyneuritis, psychoticdisorders, and seizures⁶ and have determined that it is much bettertolerated in children, as seen in a study of 67 children usingnifurtimox for Chagas disease¹¹. However in our study described indetail in Examples 13 and 14, no patients have been excluded from thestudy due to adverse drug^(5,11).

In conclusion, the examples show that Nifurtimox is cytotoxic toneuroblastoma cells. It inhibits proliferation and induces apoptosis inneuroblastoma. Apoptosis is mediated by the activation of caspase-3.Nifurtimox suppressed basal and TrkB mediated Akt phosphorylationsuggesting inhibition of TrkB signaling. Thus, nifurtimox is a potentcytotoxic and apoptotic agent and can be a therapeutic agent to combatneuroblastoma.

EXAMPLES Example 1 Reagent Preparation

Nifurtimox (from Bayer, Germany) was dissolved in dimethyl sulfoxide(DMSO) as a 20 mg/ml stock and stored in aliquots at −20° C. zVAD-fmk(Calbiochem La Jolla Calif.) was dissolved in DMSO at a concentration of10 mM and stored at −20° C. Brain-derived neurotrophic factor (BDNF) wasdissolved in sterile water (100 ng/ml) and stored at −80° C. (Santa CruzBiotechnology, Santa Cruz, Calif.). Antibodies to cleaved caspase-3 and,phosphorylated and total forms of Akt, were obtained from Cell SignalingTechnology, Beverly Mass., secondary anti-rabbit antibody coupled to HRPwas from Amersham Pharmacia Biotech, Piscataway, N.J., and propidiumIodide was from Sigma Chemical Co., St Louis, Mo.

Example 2 Cell Culture and Treatment

The human neuroblastoma cell lines IMR-32 (ATCC), SMS-KCN, SMS-KCNR(from John Maris, CHOP, Philadelphia, Pa.) were maintained in RPMI 1640media supplemented with 10% fetal bovine serum, 100 units/ml penicillinand 100 μg/ml streptomycin at 37° C. in a humidified incubator. Cellswere grown in 6 well plates or 100 mm dishes to 75% confluency and serumdeprived (RPMI 1640 with 0.1% BSA) overnight before treatment. Cellswere treated with Nifurtimox (0, 1, 10, or 20 μg/ml) for 24 hours forcaspase activation studies or for 2 hours and stimulated with 100 μg/mlBDNF (Santa Cruz Biotechnology, CA) for 10 minutes for analysis of Trksignaling.

Example 3 Nifurtimox Suppresses Akt Phosphorylation and TrkB MediatedSignaling

SMS-KCNR cells are known to express the TrkB receptor¹⁴. BDNFspecifically stimulates the TrkB receptor on neuroblastoma cells andleads to phosphorylation of AktT¹⁵ which plays a vital role in cellsurvival, ultimately leading to chemoresistance^(3,15). To understandthe signaling pathways mediating the antiproliferative and cytotoxiceffects of nifurtimox in neuroblastoma cells, TrkB and Akt signaling inSMS-KCNR cells was determined. Stimulation of SMS-KCNR cells with BDNFin the absence of nifurtimox resulted in an increased phosphorylation ofAkt (FIG. 6A, Lane 1); however, addition of nifurtimox (10 and 20 μg/ml)significantly inhibited Akt phosphorylation as indicated by decreasedband intensities (FIG. 6A, Lanes 3 and 4). Total Akt protein levels werenot altered by nifurtimox (FIG. 6A). Furthermore, nifurtimox inhibitedserum stimulated Akt phosphorylation (FIG. 6B). In the presence ofserum, there were high levels of Akt activation (FIG. 6B, Lane 1);however, addition of nifurtimox abrogated the serum stimulatedactivation of Akt. At 1.0 and 10 μg/ml doses, nifurtimox completelyinhibited the phosphorylation of. Akt (FIG. 6B, Lanes 2 and 3). Thesestudies clearly showed that nifurtimox inhibits Akt signaling inneuroblastoma. On the other hand, phosphorylation of ERK1/2 (anotherdownstream target of TrkB) was not affected. There was no change in thephosphorylation of ERK1/2 indicating that nifurtimox may not affect ERKmediated signaling (data not shown).

Example 4 Nifurtimox Suppresses the Cell Viability of NeuroblastomaCells

The growth inhibitory effect of nifurtimox on SMS-KCN, SMS-KCNR andIMR-32 neuroblastoma cell lines was determined by MTS assay. Cellviability was measured using the CellTiter 96 AQ One Solution CellProliferation Assay kit (Promega, Madison Wis.)⁴. Cells were cultured(50,000 per well) in 48 well plates for 24 hours and treated withincreasing concentrations of nifurtimox (0, 1, 10, and 20 μg/ml) for 24,48, 72 and 96 hours. Vehicle treated cells were used as control (0.001%DMSO). At the end of the incubation period, MTS reagent(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)¹²was added to each well in fresh media to a final concentration of 0.5mg/ml and incubated for 4 hours. Absorbance was measured at 490 nm usinga microplate reader (Multiskan RC, Fisher Scientific Pittsburgh, Pa.).Cell viability is represented as the mean percentage+/−SD of absorbancebefore and after treatment.

As shown in FIG. 2A, nifurtimox inhibited the growth of these cell linessignificantly in a time and dose-dependent manner and was mostpronounced in SMS-KCNR cells. The cell viability was decreased to 37%(SMS-KCNR), 59% (SMS-KCN), 78% (IMR-32) after 120 hours of treatment at20 μg/ml nifurtimox. IC₅₀ values, the concentration required to reach50% inhibition of growth, for these cell lines were determined to be14.7 μg/ml for SMS-KCNR, 25.4 μg/ml for SMS-KCN and 52.9 μg/ml forIMR-32. These studies clearly show that nifurtimox is cytotoxic toneuroblastoma cell lines. Of importance is that such cytotoxic effectsof nifurtimox are not observed with normal epithelial cells in culture

Example 5 Nifurtimox Inhibits Neuroblastoma Cell Proliferation and DNASynthesis

Cell viability is a sum total of events that include proliferation andcell death. First, we tested the ability of nifurtimox to inhibit DNAsynthesis as a measure of its effect on proliferation. DNA synthesis wasdetermined by measuring BrdU incorporation into DNA as a surrogate forproliferation.

The effect of nifurtimox on proliferation of neuroblastoma cell linesand endothelial cells was determined by BrdU (bromodeoxyuridine)incorporation assay during DNA synthesis^(6,13) following manufacturer'sinstructions (Roche Molecular Biochemicals, Indianapolis Ind.). SMS-KCNRcells (50,000 per well) were cultured in 48 well plates for 24 hours,serum deprived for 18 hours and treated with nifurtimox (0, 1, 10, and20 μg/ml) for 24 hours. Cells were labeled with 10 μM BrdU for 6 hours.The cells were fixed and the color was developed using peroxidaseconjugated anti-BrdU antibody and TMB (3,3,5,5% tetramethylbenzidine)chromogenic substrate. Absorbance was measured at 450 nm using amicroplate reader (Multiskan RC, Fisher Scientific, Pittsburgh, Pa.). Inthis assay, the color intensity correlates directly to the amount ofBrdU incorporation which in turn reflects proliferation. The results areexpressed as percentage of BrdU incorporation (+/−SD) and are shown inFIG. 2B. BrdU incorporation decreased with the increase in drugconcentration indicating that nifurtimox inhibited DNA synthesis. A 50%inhibition in DNA synthesis was observed at 18.1 μg/ml nifurtimox. BrdUincorporation studies demonstrated that nifurtimox is a potent inhibitorof neuroblastoma cell proliferation. The results are shown in FIG. 7.When treated with nifurtimox, there was a dose dependent inhibition ofDNA synthesis in HEC; a 50% inhibition was observed at 10 μg/mlnifurtimox, over 80% inhibition was observed at 20 μg/ml concentrationnifurtimox and 30% inhibition was observed at concentrations as low as 5μg/ml of nifurtimox. The BrdU incorporation studies demonstrate thatnifurtimox is a potent inhibitor of HEC proliferation.

Example 6 Nifurtimox Induces Apoptosis of Neuroblastoma Cells

The cytotoxic effect of nifurtimox in neuroblastoma cells was furtherinvestigated to understand underlying mechanisms. SMS-KCNR cells weregrown to 60% confluency in 48 well plates, incubated with increasingconcentrations of nifurtimox for 96 hours. Vehicle treated cells wereused as control. Following the treatment morphological features wereinitially observed with light microscopy using inverted microscope(Nikon Eclipse TS100, Japan) fitted with a Fuji digital camera (Japan).The cells were washed with PBS, fixed with paraformaldehyde,permeabilized with Triton X-100 at room temperature, labeled withfluorescein-12-dUTP using terminal deoxynucleotidyl transferase andcounterstained with propidium iodide (5 μg/ml). Negative controls(without terminal transferase) were included in each experiment.Apoptosis was detected by fluorescence microscopy (Nikon EclipseTE2000-E fitted with a cooled CCD camera, Japan). Propidium iodidestaining was used to detect both non-apoptotic and apoptotic cells andfluorescein staining was used to detect the apoptotic cells.

Microscopic examination of nifurtimox treated cells revealedmorphological alterations such as decreased axonal length, rounding andfloating which are suggestive of apoptosis (FIG. 3A). The morphologicalchanges were progressively pronounced with increased nifurtimoxconcentration. At 1 μg/ml nifurtimox, a decrease in axon length wasapparent when compared to vehicle treated cells (FIG. 3 A-ii and A-Irespectively). At 10 μg/ml nifurtimox, the decrease in axon length wasmore prominent and was associated with rounding of cells (FIG. 3A-iii).At 20 μg/ml nifurtimox, the cells were rounded up and floating with afew cells remained attached to the plate. (FIG. 3A-iv). Of importance isthat these morphologic apoptotic changes occurred in a dose dependentmanner.

TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine5′-triphosphate (dUTP) nick-end labeling) assay was used to confirm theinduction of apoptosis by nifurtimox. The terminal nucleotidetransferase reaction was used to identify cell nuclei containingfragmented DNA in nifurtimox treated SMS-KCNR cells (0-20 μg/ml) for 96hours. The nuclei were identified by red propidium iodide staining andTUNEL positive nuclei were identified by yellow spots in the PI andTUNEL overlay. The TUNEL assay shows a dose dependent increase inapoptosis in the SMS-KCNR cells after 72 hours of treatment withnifurtimox. With increasing drug concentration, increased apoptoticsignal is seen in the nuclei of the cells (FIG. 3B).

Example 7 Western Blot Analysis

After treatment, the cells were collected by scraping and resuspended in200 μl of E Buffer (10 mM Tris pH 7.6, 50 mM NaCl, 5 mM EDTA, 50 mM NaF,0.1 mM NaVO4, 1% Triton, 10 ug/ml Aprotinin, 10 ug/ml Leupeptin, ABSF)and incubated on ice for 20 minutes to lyse the cells. Cell lysates weresonicated for 10 seconds and centrifuged for 20 minutes at 14,000 rpm at4° C. Protein concentration was determined with BioRad proteinestimation kit (BioRad, Hercules, Calif.). Cell lysates wereelectropheresed on a 12% SDS-PAGE and blotted onto PVDF (Polyvinylidenefluoride) membranes. The blots were blocked with 5% non-fat dry milk(BioRad, Hercules, Calif.) in PBST for 1 hour. The blots were probedwith antibodies specific for cleaved caspase-3 and phosphorylated andtotal forms of Akt and ERK1/2. The protein bands were visualized usinghorseradish peroxidase-conjugated secondary antibodies(Amersham-Pharmacia Biotech, Piscataway, N.J.) followed by enhancedchemiluminescence (Upstate, Waltham, Mass.) and documented using BioRad,Gel Document System, GDS 8000 (BioRad, Hercules Calif.). Blots werestripped and probed with actin antibody as an internal loading control(Sigma Chemical Company, St. Louis, Mo.).

Example 8 BrdU Incorporation Assay of HEC

The effect of nifurtimox on proliferation was assessed by measuring BrdUincorporation during DNA synthesis. Human Endothelial Cells (HEC) (1×104cells) were cultured in 96 well plates, serum deprived for 16 hours andtreated with 0, 0.15, 0.3, 0.6125, 1.25, 2.5, 5 10 and 20 μg/ml inMedium 200 in the presence or absence of 50 ng/ml VEGF for 72 hours.BrdU (10 μM final concentration) was added to the cells and re-incubatedfor further 6 hours. The cells were fixed and the color was developedusing anti-BrdU-POD antibody and TMB chromogenic substrate. The assaywas performed according to the manufacturer's instructions. In thiscolorimetric cell proliferation assay, the color intensity correlatesdirectly to the amount of BrdU incorporated into the DNA which in turnrepresents proliferation. The results are expressed as percentage BrdUincorporation.

Example 9 Role of Caspase-3 in Apoptosis by Nifurtimox; CaspaseInhibition Studies

Confirmation that nifurtimox treatment induces apoptosis was obtained bydemonstrating activation of caspase-3, an initiator of the apoptoticcascade. SMS-KCNR cells were treated with 0, 1, 10 and 20 μg/mlnifurtimox for 24 hours and activation of caspase-3 was determined byimmunoblot analysis using antibodies that recognize activated forms ofcaspase-3. The cell viability was measured by MTS assay as above.Minimal or basal level of caspase-3 activation was observed in vehicletreated cells (FIG. 4A). Addition of nifurtimox resulted in a dosedependent and strong activation of Caspase 3 as evidenced by theappearance of 19 and 17 kDa bands (FIG. 4A). Activation of caspase-3 bynifurtimox corroborated the increase in apoptosis shown by morphologicalobservations and TUNEL assay (FIG. 3A, 3B).

To evaluate the critical role played by caspases in nifurtimox inducedapoptosis in neuroblastoma cells, Z-VAD-FMK, a pancaspase inhibitor thatirreversibly binds to the catalytic sites of caspases 1 through 9, wasused. Inactivation of caspases results in inhibition of caspase mediatedapoptosis. SMS-KCNR cells were pretreated with 50 μM pancaspaseinhibitor, Z-VAD-FMK for 90 minutes and then treated with nifurtimox (10pg/ml) for 96 hours. SMS-KCNR cells were pretreated with 50 μM Z-VAD-FMKfor 90 minutes before treatment with nifurtimox (10 μg/ml for 96 hours).This dose of 10 μg/ml nifurtimox was chosen because it is closer to IC₅₀value (FIG. 2A). The cell viability was measured by MTS assay.Nifurtimox decreased the viability of SMS-KCNR cells by 50% compared tothe vehicle treated control. However, addition of Z-VAD-FMK resulted inthe reversal of the cytotoxic activity of nifurtimox (FIG. 4B).Pretreatment with 50 μM Z-VAD-FMK for 90 minutes increased cellviability to 90% in the presence of nifurtimox. These studies clearlyshowed that the cytotoxic effect of nifurtimox was reversed bypan-caspase inhibitor, Z-VAD-FMK suggesting that the observed apoptoticeffect of nifurtimox was mediated by caspases.

Example 10 Nifurtimox Inhibits Angiogenesis—Tube Assay

First, we tested the effect of nifurtimox on tube formation on theMatrigel matrix. Stimulation with growth factors leads to morphologicaldifferentiation of endothelial cells and formation of tube likestructures on matrices. The endothelial tube formation assay wasperformed in Matrigel bed. Briefly, Matrigel bed was prepared by usingpre-cooled pipettes, plates and tubes on ice. Growth factor-reducedMatrigel was thawed overnight at 4° C. and mixed to homogeneity, Cultureplates (48-well) were coated with 0.1 ml of Matrigel and allowed togelatinize at 37° C. for 30 minutes. HEC (2×10⁴) per well were seeded onthe Matrigel bed and cultured in EBM-2 basal media containing nifurtimox(0, 10 or 20 μg/ml) in the presence or absence of VEGF (50 ng/ml) for 8hours. Capillary networks were photographed using a phase-contrastmicroscope (Nikon Eclipse TS100, fitted with Fuji digital camera,Japan), and the number of tubes was quantified by counting the branchingpoints from four quadrants of each well.

On Matrigel substratum, endothelial cells form aggregates, and thenaggregated cells start to sprout and fuse to form tube-like structures.In the presence of growth factor HEC formed organized elongatedtube-like structures resembling capillaries with an extensive network.(FIG. 8A). However, in the absence of growth factor (control), no suchorganized structures were observed (FIG. 8B). In the presence of VEGF,nifurtimox exhibited a marked inhibitory effect on the formation oftube-like structures by HEC. Tube formation was diminished withformation of incomplete network of capillary like structure (FIG. 8C).Nifurtimox showed 50% (±7%) inhibition of tube formation at 20 μg/mlrespectively. These findings suggest that nifurtimox inhibits the tubeformation step in angiogenesis.

Example 11 Nifurtimox Inhibits Angiogenesis—Aortic Ring Assay

Next, we tested the ability of nifurtimox to inhibit growth factorinduced aortic capillary sprouts using rat aortic rings (explants)embedded in Matrigel beds. Matrigel bed mimics physiologicalextracellular matrix representing its natural composition andarchitecture. Due to these features, Matrigel enables several celltypes, including endo-thelial cells, to maintain in culture their invivo phenotype and 3-dimensional organization. Aortic arches wereremoved from euthanized rats and immediately transferred to a culturedish containing ice-cold serum-free media. The peri-aortic fibroadiposetissue was carefully removed with fine microdissection forceps andiridectomy scissors paying special attention not to damage the aorticwall. Aortic rings (1 mm thick) were sectioned and extensively rinsed in5 consecutive washes of Medium 200. Ring shaped explants of rat aortawere then embedded in Matrigel beds in 48 well plates, treated with 1,10 or 20 μg/ml nifurtimox in the presence or absence of growth factorsand incubated at 37° C. in a tissue culture incubator. The explants wereexamined every second day with a Nikon Eclipse TS100 inverted microscopeat an appropriate magnification and photographed at the end of 9th day.On the ninth day, the capillary sprouting were quantified by grading andrecording the extent of sprouting directly reflecting angiogenesis usingNikon Eclipse TS100 fluorescent microscope (Japan) and Fuji digitalcamera.

The aortic rings cultured in serum free media showed little or nosprouting (FIG. 7A). When the aortic rings were cultured in the presenceof growth factors (complete media), sprouting of microvessels wereinitially noticed in on day 3-4, with the number and length ofmicrovessels increasing with prolonged culture time. Microvesseloutgrowths arose from the edges of the implanted ring. The initiallylinear sprouts of HEC progressively branched, anatomized, and gave riseto a complex microvascular network. A thick capillary network ofbranching microvessels with tubes and loops developed from the peripheryof the explant, which was found to spread towards the edges of the well(FIG. 7B). In contrast, this microvessel formation was dramaticallydecreased in a dose dependent manner when nifurtimox was added tocomplete media. A marked delay in outgrowth of the sprouts from theexplants with a regression in both the number of microvessels and thenumber of branches was observed in nifurtimox treated aortic rings. Atlower doses (1 and 10 μg/ml) of nifurtimox, the capillary network wassparse and incomplete (FIGS. 7C and D) and at higher doses of nifurtimox(20 μg/ml) there was complete inhibition of micro-vascular sprouting(FIG. 7E). The endothelial nature of the microvessels was demonstratedby staining the aortic rings with Dil-Ac-LDL (Data not shown).Dil-Ac-LDL is selectively taken up by endothelial cells and does notimpair their growth, survival or functions. In accordance with theirendothelial origin, the tubular outgrowths were fluorescent andcorresponds to the phase contrast micrographs. Analysis of theout-growing microvessels revealed that nifurtimox strongly inhibitedangiogenesis.

Example 12 Decrease in Tumor Size in Mice

Xenograft experiments using 10⁷ SMS-KCNR cells injected into nude miceand treated with or without 150 mg/kg/day of nifurtimox in food pelletsfor 30 consecutive days. Upon harvest, tumor size was measured. Theresults are shown in FIG. 11. A greater than three fold decrease intumor size was seen (1.14 grams versus 0.3 grams).

Example 13 Treatment of a Human Patient Having Neuroblastoma and ChagasDisease

A five year old female patient with progressive refractory neuroblastomabeing treated with the conventional chemotherapeutics Cyclophosphamide(250 mg/m²/dose in 50 ml/m² NS, infused over 30 minute) and Topotecan(0.75 mg/m²/dose in 50 ml NS or D5W, infused over 30 minutes) acquiredChagas disease from a blood transfusion. Chagas disease is a parasiticdisease caused by Trypanasoma cruzi endemic to South America. Althoughnifurtimox is not currently approved by FDA in the US for Chagas or anyother diseases, it was obtained through the Center for Disease Control(CDC) in order to treat the Chagas disease in the patient.

The patient was started on a 15-20 mg/kg/day dose of Nifurtimox, by oraltablet three times daily; and the patient's tumor subsequentlyregressed'. When in remission and off all medications the patientrelapsed, was restarted on nifurtimox, cyclyphosphamide and topotecan,and again demonstrated tumor regression. There were no significant sideeffects or evidence of significant organ toxicity in this patient.

Example 14 Treatment of Human Patients Having Neuroblastoma Only

Three additional patients with relapsed neuroblastoma (but not withChagas disease) were treated with a 20 mg/kg/day (in three doses) ofNifurtimox alone or in combination with additional treatments asdescribed below. All showed tumor regression and tolerated theirtreatment regimen well, with no significant side effects or evidence oforgan toxicity.

Age Ascorbic Acid Cyclophosphamide Topotecan Patient 4 none Beginningday 15, Beginning day 15, A 250 mg/m²/dose in 50 0.75 mg/m²/dose in 50ml ml/m² NS, infused over NS or D5W, infused over 30 minute; daily for 530 minutes; daily for 5 consecutive days every consecutive days every 2121 days, for 3 cycles days, for 3 cycles Patient 6 5 grams twice 250mg/m²/dose in 50 0.75 mg/m²/dose in 50 ml B weekly, I.V. ml/m² NS,infused over NS or D5W, infused over increased up to 30 minute; dailyfor 5 30 minutes; daily for 5 25 grams twice consecutive days everyconsecutive days every 21 weekly 21 days, for 3 cycles days, for 3cycles Patient 9 Beginning day 250 mg/m²/dose in 50 0.75 mg/m²/dose in50 ml C 22, 5 grams ml/m² NS, infused over NS or D5W, infused over twiceweekly 30 minute; daily for 5 30 minutes; daily for 5, increased up toconsecutive days every consecutive days every 21 30 grams twice 21 days,for 3 cycles days, for 3 cycles weekly

Patient A presented with multiply relapsed neuroblastoma and was startedon Nifurtimox at the dosage described above. After two weeks thepatient's quality of life had improved and tumor stabilization wasobserved. After four months of treatment with Nifurtimox co-administeredwith cyclophosphamide and topotecan, significant and continuous tumorregression was observed. After two months on the treatment regimen,Patient B had normalization of tumor markers; bone marrow aspirate wasclear and negative for disease and MIBG scan, which had multiple spotsupon presentation, showed one spot remaining. Patient C had an initialtumor marker VMA of 22 and bone marrow positive for tumor. By Day 21prior to the start of ascorbic acid co-administration, the patient'stumor marker VMA bad decreased to 17 and the patient's bone marrow wasnegative. After 3 cycles of treatment, the patient's tumor marker VMAhad further decreased to 13.

Example 15 Dose Toxicity Studies in Human Patients

The standard therapeutic dose in children for the treatment of Chagasdisease is 15-20 mg/kg/day. Toxicity at these doses is generally mild.For dose toxicity studies, escalating dosages as set forth in thefollowing table are used.

Dose Escalation Level Number of Patients/group Dose (mg/kg/day) 1 3 20 23 30 3 3 40 4 3 50 5 3 60

On days 1-21, the patients are treated with Nifurtimox alone in oraldosages three times per day. Thereafter, Cyclophosphamide and topotecanare added to the Nifurtimox treatment regimen Each cycle of chemotherapyis given over 5 consecutive days every 21 days as follows: Prehydrationwith 500 ml/m² D5W ½ NS over 30-60 minutes, along with antiemetictherapy. Cyclophosphamide, 250 mg/m²/dose in 50 ml/m² NS, infused over30 minute. Topotecan, 0.75 mg/m²/dose in 50 ml NS or D5W, infused over30 minutes. FILGRASTIM, 5 micrograms/kg sc daily is given beginning24-48 hours after the completion of day 5 chemotherapy and untilneutrophil recovery. PEG-filgrastim 6 mg sc may be substituted forpatients >40 kg at the discretion of the treating physician.

This 21 day cycle is repeated for a total of 3 times with re-evaluationprior to each cycle. Re-evaluation will include CBC, LDH, ferritin,urine catecholamines, MRI of primary site, and MIBG scan, plus bonemarrow aspiration/biopsy if positive at beginning of protocol. Treatmentbeyond the 4 cycles of therapy is left up to the discretion of thetreating physician.

Example 16 Improved Synthesis of Nitrofurans (Specifically Nifurtimox)

An improved, more efficient, and less hazardous synthesis of thenitrofuran side chain was accomplished and is illustrated in FIG. 12 anddescribed below.

Referring to FIG. 12, the synthesis of diol (1) was achieved with basepromoted nucleophilic addition of mercaptoethanol to propylene oxide atreflux in ethanol as solvent. As known by those skilled in the art, thisreaction is achievable with solvents other than ethanol and with basesother than sodium ethoxide.

¹H NMR (CDCl₃): δ 3.868 (1H), 3.734 (2H), 2.725 (4H), 2.465 (2H), 1.222(3H) MS (FAB): 159 [H+Na]⁺

The synthesis of sulfone diol (2) was achieved with hydrogen peroxidesolution under catalytic amounts of acids, such as phosphoric acid, atreflux. The solvent was removed under reduced pressure or under streamof air, N₂ or argon and then dried under high vacuum.

¹H NMR (CDCl₃): δ 4.48 (1H), 4.12 (2H), 3.31 (3H), 3.13 (1H), 1.31 (3H);MS (FAB):191 [M+Na]⁺.

The cyclization of sulfone diol (2) (1 eq.) to compound (3) was achievedby base promoted hydrazine insertion with tBoc-NHNH₂ (1.25 eq) at refluxovernight. The reaction mixture was extracted with ethyl acetate andconcentrated under reduced pressure to afford a semi solid product.

MS (FAB): 165.2 [M+H]⁺, 187 [M+Na]⁺.

The synthesis of Nifurtimox was achieved by condensation of compound (3)(1.1 eq.) with 5-nitro-2-furaldehyde (1 eq.) at reflux, for periods ofup to 24 hrs, in alcoholic solvents such as ethyl alcohol, preferablyanhydrous. The reaction mixture was filtered hot or cold under suctionand afforded the desired product.

¹H NMR (CDCl₃+CD₃OD): δ 7.40 (1H), 7.32 (1H), 6.67 (1H), 4.16-4.23 (1H),3.98-4.04 (1H), 3.64-3.74 (1H), 2.80-3.02 (4H) and 1.45-1.47 (d, 3H)

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of thedisclosure. Although the compositions and methods of the invention havebeen described in terms of preferred embodiments, it will be apparent tothose having ordinary skill in the art that variation may be made to thecompositions and methods without departing from the concept, spirit andscope of the invention. For example, certain agents and composition thatare chemically related may be substituted for the agents describedherein if the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

All publications, patent applications, patents and other documents citedherein are incorporated by reference in their entirety. In case ofconflict, this specification including definitions will control. Inaddition the material, methods and examples are illustrative only andnot intended to be limiting.

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1. Use of a nitrofuran compound for preparing a medicament for thetreatment of cancer or the inhibition of angiogenesis in a mammal. 2.Use according to claim 1, wherein the cancer is neuroblastoma,medulloblastoma, peripheral malignant nerve sheath tumor, ependymoma,chraniopharyngioma, astrocytoma, meningioma, germinoma, glioma, mixedglioma, choroid plexus tumor, oligodendroglioma, peripheralneuroectodermal tumor, primitive neuroectodermal tumor (PNET), CNSlymphoma, pituitary adenoma, or Schwannoma.
 3. Use according to claim 2,wherein the cancer is neuroblastoma.
 4. Use according to claim 1,wherein the nitrofuran compound is Nifurtimox, Furazolidine orNifuratel, and/or an analog thereof.
 5. Use according to claim 4,wherein the nitrofuran or nitrofuran is Nifurtimox.
 6. Use according toclaim 1, wherein the medicament is in a form suitable for administrationorally, intrathecally, intracranially or intramuscularly.
 7. Useaccording to claim 1, wherein the nitrofuran compound, or theNifurtimox, Furazolidine, Nifuratel or analog thereof, is present in themedicament in an amount to give a unit dosage of 200-300 mg.
 8. Useaccording to claim 7 wherein the medicament is to be administered atintervals ranging from once a day to once three times a day.
 9. Useaccording to claim 8 wherein the medicament is to be administered threetimes per day for three weeks.
 10. Use according to claim 1, wherein themedicament comprises a therapeutically effective and physiologicallyacceptable amount of at least one other active ingredient or agent. 11.Use according to claim 10, wherein the at least one other activeingredient is ascorbic acid or buthionine sulfoximine.
 12. Apharmaceutical unit dosage form comprising an amount of a nitrofurancompound effective to treat neuroblastoma or other central nervoussystem cancer.
 13. The pharmaceutical unit dosage form according toclaim 12, wherein the unit dosage is 200-300 mg.
 14. The pharmaceuticalunit dosage form according to claim 13, formulated for oral,intrathecal, intracranial, intravenous, or intramuscular administration.15. The pharmaceutical unit dosage form according to claim 14 furthercomprising ascorbic acid or buthionine sulfoximine.
 16. A kit comprisinga medicament for the treatment of neuroblastoma or other central nervoussystem cancer comprising nifurtimox formulated for oral, intrathecal,intracranial, intravenous, or intramuscular administration.
 17. The kitaccording to claim 16, wherein the medicament comprisise atherapeutically effective and physiologically acceptable amount of atleast one other active ingredient or agent.
 18. The kit according toclaim 16, additionally including instructions for use of the medicament.19. A method of making nifurtimox comprising (a) combiningmercaptoethanol and propylene oxide under suitable conditions and in thepresence of suitable solvents so as to achieve diol formation at reflux;(b) reacting the diol formed in step (a) with a hydrogen peroxidesolution under a catalytic amount of an acid at reflux to form thesulfone diol; (c) reacting the sulfone diol formed in step (b) withIBoC—NHNH₂ at reflux overnight, extracting the reaction mixture, andconcentrating the mixture under reduced pressure to afford a semi-solidproduct; (d) condensing the product formed in step (c) with5-nitro-2-furaldehyde at reflux in an anhydrous alcoholic solvent andfiltering the reaction mixture under suction.